Humanized tau antibodies in a alzheimer&#39;s disease

ABSTRACT

The present invention is in the fields of biochemistry, molecular biology, and Alzheimer&#39;s disease diagnosis, prevention, and treatment Provided herein are humanized antibodies against human tau that are capable of discriminating between normal (healthy) and pathological (disease-associated) tau.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase application based onPCT/IB2015/002610, filed Nov. 18, 2015, which claims the benefit ofpriority of United States Provisional Application No. 62/081,809, filedNov. 19, 2014, the content of both of which is incorporated herein byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 19, 2014, isnamed 11634.6003.00000_SL.txt and is 284,497 bytes in size.

FIELD

The present invention is in the fields of biochemistry, molecularbiology, and Alzheimer's disease diagnosis, prevention, and treatment.Provided herein are humanized antibodies against human tau that arecapable of discriminating between normal (healthy) and pathological(disease-associated) tau.

BACKGROUND

Alzheimer's disease (AD) is a progressive neurodegenerative disorderthat destroys higher brain structures, such as those involved in memoryand cognition. The disease leads to deficits in cognitive function anddeclines in memory, learning, language, and in the ability to performintentional and purposeful movements. There is a need for effectivemethods and compositions for treatment and prophylaxis of AD.

AD is histologically characterized by the presence of extraneuronalplaques and intracellular and extracellular neurofibrillary tangles inthe brain. Plaques are composed mainly of β amyloid (Aβ), whereastangles comprise pathological forms of tau, such as pathological tauconformers and their aggregates. A recognized role for tau in ADpathology has been demonstrated in numerous studies. For example, Braakshowed that the closest correlate for AD neurodegeneration was thepresence of tau tangles, and not of amyloid plaques (Braak, H., et al.Neuropathological staging of Alzheimer-related changes. Acta Neuropathol82:239-259 (1991)).

Tau belongs to a family of intrinsically disordered proteins,characterized by the absence of a rigid three-dimensional structure intheir physiological environment (Skrabana et al., 2006). However, tautruncation and hyperphosphorylation can cause pathologicaltransformations from an intrinsically disordered state to multiplesoluble and insoluble misdisordered structures, including paired helicalfilaments (PHFs) and other aggregates (Wischik, C. M., Novak, M.,Edwards, P. C., Klug, A., Tichelaar, W., Crowther, R. A. (1988).Structural characterization of the core of the paired helical filamentof Alzheimer disease, Proc Natl Acad Sci USA 85, 4884-8; Wischik, C. M.,Novak, M., Thøgersen, H. C., Edwards, P. C., Runswick, M. J., Jakes, R.,Walker, J. E., Milstein, C., Roth, M., Klug, A. (1988), Isolation of afragment of tau derived from the core of the paired helical filament ofAlzheimer disease, Proc Natl Acad Sci USA 85, 4506-10; Novak et al.,1993; Skrabana et al., 2006; Zilka, N., et al. Chaperone-like AntibodiesTargeting Misfolded Tau Protein: New Vistas in the Immunotherapy ofNeurodegenerative Foldopathies. Journal of Alzheimer's disease 15 (2008)169-179; Kovacech B, Novak M. (2010). Tau truncation is a productiveposttranslational modification of neurofibrillary degeneration inAlzheimer's disease. Curr Alzheimer Res December; 7(8):708-16); KovacechB, Skrabana R, Novak M. (2010). Transition of tau protein fromdisordered to misordered in Alzheimer's disease, Neurodegener Dis 7:24-27). These structural changes lead to a toxic gain of function, to aloss of physiological function of the native protein, or both (Zilka etal., 2008; Kovacech B, Novak M. (2010). Tau truncation is a productiveposttranslational modification of neurofibrillary degeneration inAlzheimer's disease. Curr Alzheimer Res December; 7(8):708-16); KovacechB, Skrabana R, Novak M. (2010), Transition of tau protein fromdisordered to misordered in Alzheimer's disease. Neurodegener Dis 7:24-27).).

Tau's physiological function is in mediating the assembly of tubulinmonomers into microtubules that constitute the neuronal microtubulesnetwork (Buee, L., Bussiere, T., Buee-Scherrer, V., Delacourte, A., Hof,P. R. (2000). Tau protein isoforms, phosphorylation and role inneurodegenerative disorders. Brain Research. Brain Research Reviews. 33,95-130). Tau binds to microtubules through repetitive regions located inthe C-terminal portion of the protein. Butner K A, Kirschner M W. 1991.Tau protein binds to microtubules through a flexible array ofdistributed weak sites. J Cell Biol 115: 717-730; Lee G, Neve R L, KosikK S. 1989. The microtubule binding domain of tau protein. Neuron 2:1615-1624. These repeat domains (R1-R4), are not identical to eachother, but comprise highly conserved 31-32 amino acids (Taniguchi T,Surnida M, Hiraoka S, Tomoo K, Kakehi T, Minoura K, Sugiyama S, Inaka K,Ishida T, Saito N, Tanaka C 2005 (Effects of different anti-tauantibodies on tau fibrillogenesis: RTA-1 and RTA-2 counteract tauaggregation. FEBS Lett 579:1399-1404; Taniguchi S, Suzuki N, Masuda M,Hisanaga S, Iwatsubo T, Goedert M, Hasegawa M. Inhibition ofheparin-induced tau filament formation by phenothiazines, polyphenols,and porphyrins, J Biol Chem 280:7614-7623 (2005)). In the human brain,there are six unique isoforms of tau, which differ from each other inthe presence or absence of certain amino acids in the N-terminal portionof tau, in combination with either three (R1, R3, and R4) or four(R1-R4) repeat domains, at the C-terminal end of the protein. See alsoFIG. 1, which shows the six human isoforms (2N4R, 1N4R, 2N3R, 0N4R,1N3R, and 0N3R SEQ ID Nos. 151-156, respectively, in order ofappearance).). It has been proposed that the most potent part of tau toinduce microtubule polymerization are the sequences 306-VQIVYK-311 (SEQID NO: 146) and 274-KVQIINKK-281 region (SEQ ID NO: 144), overlappingR1-R2. (von Bergen M, Friedhoff P, Biernat J, Heberle J, Mandelkow E M,Mandelkow E. 2000. Assembly of tau protein into Alzheimer paired helicalfilaments depends on a local sequence motif ((306)VQIVYK(311)) formingbeta structure. Proc Natl Acad Sci USA 97: 5129-5134.)Id.

In addition, tau's pathological and physiological functions appear to beinfluenced by the specific structural conformation, and theintrinsically disordered structure, adopted by the full length proteinisoforms and their fragments. For example, Kontsekova et al. described aconformational region (encompassing residues297-IKHVPGGGSVQIVYKPVDLSKVTSKCGSL-325 (SEQ ID NO: 145) within certaintruncated tau molecules which had a significant relationship to thefunction of those truncated tau molecules on microtubule assembly (WO2004/007547).

In addition to their physiological role, tau repeats are believed toparticipate in the formation of pathological tau aggregates and otherstructures. Thus, there is a need for tau-targeted therapeutic anddiagnostic approaches that are capable of discriminating betweenphysiological and pathological microtubule binding repeatregion-mediated activities. For example, the pronase resistant core ofpathological paired helical filaments (PHFs) consists of the microtubulebinding regions of 3- and 4-repeat tau isoforms (Jakes, R., Novak, M.,Davison, M., Wischik, C. M. (1991).

Identification of 3- and 4-repeat tau isoforms within the PHF inAlzheimer's disease. EMBO J 10, 2725-2729; Wischik, et al. 1988a;Wischik, et al. 1988b). Further, Novak et al. showed that the proteaseresistant core of the PHFs, which is 93-95 amino acids long, wasrestricted to three tandem repeats (Novak, M., Kabat, J., Wischik, C. M.(1993). Molecular characterization of the minimal protease resistant tauunit of the Alzheimer's disease paired helical filament. EMBO J 12,365-70). Von Bergen et al. determined a minimal-tau peptide/interactionmotif (306-VQIVYK-311; SEQ ID NO: 146), as well as a second site on tau(275-VQIINK-280) (SEQ ID NO: 147), which form beta-sheets and aredescribed as potentially responsible for initiating the formation ofPHFs, a pathological tau aggregate (von Bergen M, Friedhoff P, BiernatJ, Heberle J, Mandelkow E M, Mandelkow E. 2000. Assembly of tau proteininto Alzheimer paired helical filaments depends on a local sequencemotif ((306)VQIVYK(311)) forming beta structure. Proc Natl Acad Sci USA97: 5129-5134); EP 1214598; WO 2001/18546). See FIG. 2 for a functionalmap of tau. Consequently, current strategies aim at generatinganti-aggregating drugs that do not disrupt tau's intracellular role inmicrotubule stabilization.

Moreover, while under physiological circumstances tau is considered anintracellular cytoplasmic protein, intracellular tau can be releasedinto the extracellular space and contribute to neurodegeneration(Gómez-Ramos, A., Diaz-Hernández, M., Cuadros, R., Hernández, F., andAvila, J. (2006). Extracellular tau is toxic to neuronal cells. FEBSLett 580(20), 4842-50). Indeed, neuronal loss has been linked to thetopographic distribution of neurofibrillary tangles (made up of tauprotein) in AD brains (West, M. J., Coleman, P. D., Flood, D. G.,Troncoso, J. C. (1994). Differences in the pattern of hippocampalneuronal loss in normal aging and Alzheimer's disease. Lancet 344,769-72; Gómez-Isla, T., Price, J. L., McKeel Jr, D. W., Morris, J. C.,Growdon, J. H., Hyman, B. T. (1996). Profound loss of layer IIentorhinal cortex neurons occurs in very mild Alzheimer's disease. JNeurosci 16(14), 4491-500; Gomez-Isla T, Hollister R, West H, Mui S,Growdon J H, Petersen R C, Parisi J E, Hyman B T. Neuronal losscorrelates with but exceeds neurofibrillary tangles in Alzheimer'sdisease. Ann Neurol 41:17-24 (1997)). Further, the levels of total tauand phosphorylated tau are increased in the cerebrospinal fluid (CSF) ofpatients with AD (Hampel, H., Blennow, K., Shaw, L. M., Hoessler, Y. C.,Zetterberg, H., Trojanowski, J. Q. (2010). Total and phosphorylated tauprotein as biological markers of Alzheimer's disease. Exp Gerontol45(1), 30-40), and extracellular tau has been described as “ghosttangles” in the brain (Frost, B., Diamond, M. I. (2009). The expandingrealm of prion phenomena in neurodegenerative disease. Prion 3(2):74-7),indicating that intracellular tau is released into extracellular space.In addition, extracellular tau aggregates can enter cells and stimulatefibrillization of intracellular tau, further seeding tau monomer forproduction of pathological tau aggregates (Frost et al., 2009). Suchstudies have highlighted that extracellular, insoluble tau could act asa transmissible agent to spread tau pathology throughout the brain in aprion-like fashion (Frost, B., Jacks, R. L., Diamond, M. I. (2009).Propagation of tau misfolding from the outside to the inside of a cell.J Biol Chem 284(19), 12845-52; Frost et al., 2009; Frost, B., Diamond,M. I. (2009). The expanding realm of prion phenomena inneurodegenerative disease. Prion 3(2):74-7). Targeting abnormal tau canreduce tau-associated extracellular and intracellular pathology. See,Eva Kontsekova, Norbert Zilka, Branislav Kovacech, Petr Novak, MichalNovak. 2014, First-in-man tau vaccine targeting structural determinantsessential for pathological tau-tau interaction reduces tauoligomerisation and neurofibrillary degeneration in an Alzheimer'sdisease model. Alzheimer's Research & Therapy, 6:44. Therefore, there isa need for treatments capable of decreasing extracellular tau, either byimpeding its formation, promoting its clearance, or both, as well as fortreatments that decrease intracellular disease tau. An increasedunderstanding of the molecular mechanisms underlying the pathologicaltransformations of tau has opened up the possibility of specificallytargeting pathological modifications of tau for therapeutic purposes.

International Publication No. WO2013/041962 by Novak et al. describesthe discovery of four regions of tau that promote tau-tau aggregation inAD and antibodies that prevent tau aggregation by binding to those fourregions.

Although other studies have described antibodies that bind to tausequences, and some of those antibodies also reportedly interfere withtau aggregation and clearance (Asuni A A, Boutajangout A, Quartermain D,Sigurdsson E M. Immunotherapy targeting pathological tau conformers in atangle mouse model reduces brain pathology with associated functionalimprovements. J Neurosci 27:9115-9129 (2007)), no monoclonal anti-tauantibody is yet reportedly undergoing clinical trials in AD.

The success of foreign (mouse) monoclonal antibodies in human treatmenthas been, in part, impeded by immunogenic antiglobulin responses mountedby the human recipient against such foreign therapeutics. Thesecomplicate both the safety and pharmacokinetic properties of antibodies.These challenges have led to the development of engineered antibodiesthat carry a lower risk of immune reactions. A variety of patentedengineering technologies (e.g., chimerization, humanization, CDRgrafting, framework grafting, affinity maturation, phage display,transgenic mice) are constantly being developed to facilitate thisprocess. For recent review, see Safdari Y1, Farajnia S, Asgharzadeh M,Khalili M. Antibody humanization methods—a review and update. 2013.Biotechnol Genet Eng Rev. 29:175-86. doi:10.1080/02648725.2013.801235and Almagro JC1, Fransson J. Humanization of antibodies. 2008. FrontBiosci. 13:1619-33.

Humanized antibodies are designed, primarily, to retain the specificityand affinity of the parent antibody while having human constant regions,which ideally would present less of an immunogenic target to thepatient. The typical humanized antibody carries the complementaritydetermining regions (CDRs) of a parent antibody of mouse or rat origin,and framework (FR) and constant regions that are mostly of human originbut have often been mutated to retain the parent antibody's bindingproperties. But antigen-binding affinity and specificity are not theonly factors affecting the biological activity and clinical success ofan antibody. Improving an antibody's activation of the patient's immunesystem is key to the value of some humanized antibodies, whereas forothers reduction of cellular-mediated toxicity is a goal. Ultimately, anincreased understanding of antibody structure and activity allowsresearchers to engineer, often through mutations, more advancedhumanized antibodies that are more homogeneous with better antigenbinding properties (binding affinity, target specificity), effectorfunctions, stability, expression level, purification properties,pharmacokinetics, and pharmacodynamics. Many of these improvements areimportant for the commercial viability of a given antibody. Sometimes,after target binding affinity and specificity is achieved, it isnecessary to mutate some of the amino acids in the CDRs or FRs todecrease a humanized antibody's susceptibility to aggregation. Othertimes, the constant regions are altered (switched or mutated) forimproved effector functions. These and other aspects of antibodyfunction and activity continue to present challenges to the developmentof antibodies for clinical use. Described below is a set of humanizedantibodies against tau that have been engineered to possess unexpectedadvantageous properties. Also provided below are novel methods andcompositions comprising these highly specific and highly effectiveantibodies having the ability to specifically recognize and bind topathological tau, impeding its aggregation. All these antibodies,methods, and compositions are useful for diagnosis and treatment of ADand related tauopathies.

SUMMARY OF THE INVENTION

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiment and the appended claims.

Disclosed herein is a humanized anti-tau antibody, or a tau-bindingfragment thereof, wherein said antibody or binding fragment comprises:

a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 ofSEQ ID NOs. 1, 2, 3, respectively, and a framework from humanimmunoglobulin M65092 (SEQ ID NO. 71;

a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 ofSEQ ID NOs. 4, 5, 6, respectively, and a framework from humanimmunoglobulin X72449 (SEQ ID NO. 65; and

heavy chain and light chain constant regions each from a humanimmunoglobulin; and wherein said heavy chain framework has beensubstituted at one or more of positions selected from 9, 21, 27, 28, 30,38, 48, 67, 68, 70, and 95; said light chain framework has either notbeen substituted or has been substituted at position 5; and wherein saidpositions are according to Kabat. In some embodiments, the antibodybinds one or two epitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG(SEQ ID NO: 149), and HKPGGG (SEQ ID NO: 150). In some embodiments, theantibody binds all three epitopes.

Such antibody and binding fragment is also contemplated in a form,wherein heavy chain position 9 is occupied by P, position 21 is occupiedby P, position 27 is occupied by Y, position 28 is occupied by I,position 30 is occupied by T, position 38 is occupied by K, position 48is occupied by I, position 67 is occupied by K, position 68 is occupiedby A, position 70 is occupied by L, and/or position 95 is occupied by F.In one embodiment, the light chain position 5 is occupied by S. In someembodiments, only two of these 11 positions are occupied as such. Insome embodiments, only three of these 11 positions are occupied as such.In some embodiments, only four of these 11 positions are occupied assuch. In some embodiments, only five of these 11 positions are occupiedas such. In some embodiments, only six of these 11 positions areoccupied as such. In some embodiments, only seven of these 11 positionsare occupied as such. In some embodiments, only eight of these 11positions are occupied as such. In some embodiments, only nine of these11 positions are occupied as such. In some embodiments, only ten ofthese 11 positions are occupied as such. In some embodiments, all 11 ofthese 11 positions are occupied as such. In some embodiments, theantibody binds one or two epitopes chosen from HQPGGG (SEQ ID NO: 148),HVPGGG (SEQ ID NO: 149), and HKPGGG (SEQ ID NO: 150). In someembodiments, the antibody binds all three epitopes.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is selected fromthat of RHA through RHM, SEQ ID NOs. 13-25, respectively; and thesequence of the light chain variable region is SEQ ID NO. 26 (RKA).

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is selected fromthat of RHA through RHM SEQ ID NOs. 13-25, respectively; and thesequence of the light chain variable region is SEQ ID NO. 27 (RKB).

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 13,RHA, and the sequence of the light chain variable region is SEQ ID NO.26, RKA.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, and the sequence of the light chain variable region is SEQ ID NO.26, RKA.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 15,RHC, and the sequence of the light chain variable region is SEQ ID NO.26, RKA.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 16,RHD, and the sequence of the light chain variable region is SEQ ID NO.26, RKA.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, and the sequence of the light chain variable region is SEQ ID NO.26, RKA.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 18,RHE, and the sequence of the light chain variable region is SEQ ID NO.26, RKA.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 19,RHG, and the sequence of the light chain variable region is SEQ ID NO.26, RKA.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 20,RHH, and the sequence of the light chain variable region is SEQ ID NO.26, RKA.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 21,RHI, and the sequence of the light chain variable region is SEQ ID NO.26, RKA.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 22,RHJ, and the sequence of the light chain variable region is SEQ ID NO.26, RKA.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 23,RHK, and the sequence of the light chain variable region is SEQ ID NO.26, RKA.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 24,RHL, and the sequence of the light chain variable region is SEQ ID NO.26, RKA.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 25,RHM, and the sequence of the light chain variable region is SEQ ID NO.26, RKA.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 13,RHA, and the sequence of the light chain variable region is SEQ ID NO.27, RKB.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, and the sequence of the light chain variable region is SEQ ID NO.27, RKB.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 15,RHC, and the sequence of the light chain variable region is SEQ ID NO.27, RKB.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 16,RHD, and the sequence of the light chain variable region is SEQ ID NO.27, RKB.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, and the sequence of the light chain variable region is SEQ ID NO.27 RKB.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 18,RHF, and the sequence of the light chain variable region is SEQ ID NO.27, RKB.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 19,RHG, and the sequence of the light chain variable region is SEQ ID NO.27, RKB.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 20,RHH, and the sequence of the light chain variable region is SEQ ID NO.27, RKB.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 21,RHI, and the sequence of the light chain variable region is SEQ ID NO.27, RKB.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 22,RHJ, and the sequence of the light chain variable region is SEQ ID NO.27, RKB.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 23,RHK, and the sequence of the light chain variable region is SEQ ID NO.27, RKB.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 24,RHL, and the sequence of the light chain variable region is SEQ ID NO.27, RKB.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 25,RHM, and the sequence of the light chain variable region is SEQ ID NO.27, RKB.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, the sequence of the light chain variable region is SEQ ID NO. 26,RKA, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 16,RHD, the sequence of the light chain variable region is SEQ ID NO. 26,RKA, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, the sequence of the light chain variable region is SEQ ID NO. 26,RKA, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 25,RHM, the sequence of the light chain variable region is SEQ ID NO. 26,RKA, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 26,RHD, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 16,RHD, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

Also contemplated is a humanized anti-tau antibody, or a tau-bindingfragment thereof, wherein said antibody or binding fragment comprises:

a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 ofSEQ ID NOs. 1, 2, 3, respectively, and a framework from humanimmunoglobulin M65092 (SEQ ID NO. 71); and

a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 ofSEQ ID NOs. 4, 5, 6, respectively, and a framework from humanimmunoglobulin X72449 (SEQ ID NO. 65); and

heavy chain and light chain constant regions from a humanimmunoglobulin, preferably IgG1 or IgG4.

Further contemplated is an anti-tau antibody, or a tau-binding fragmentthereof, wherein said antibody or binding fragment comprises thefollowing complete chains:

a heavy chain having the amino acid sequence of any one of SEQ ID NO.28-40; and

a light chain variable domain having the amino acid sequence of any oneof SEQ ID NO. 26 and 27. In some embodiments, the antibody binds one ortwo epitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO:149), and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibodybinds all three epitopes.

Further contemplated is an anti-tau antibody, or a tau-binding fragmentthereof, wherein said antibody or binding fragment comprises thefollowing complete chains:

a heavy chain having the amino acid sequence of any one of SEQ ID NO.43-55; and

a light chain variable domain having the amino acid sequence of any oneof SEQ ID NO. 26 and 27. In some embodiments, the antibody binds one ortwo epitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO:149), and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibodybinds all three epitopes.

Further contemplated is an anti-tau antibody, or a tau-binding fragmentthereof, wherein said antibody or binding fragment comprises thefollowing complete chains:

a heavy chain having the amino acid sequence of any one of SEQ ID NO.43-55; and

a light chain variable domain having the amino acid sequence of any oneof SEQ ID NO. 27 and 58. In some embodiments, the antibody binds one ortwo epitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO:149), and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibodybinds all three epitopes.

Further contemplated is an anti-tau antibody, or a tau-binding fragmentthereof, wherein said antibody or binding fragment comprises thefollowing complete chains:

a heavy chain having the amino acid sequence of SEQ ID NO. 31; and

a light chain variable domain having the amino acid sequence of SEQ IDNO. 57. In some embodiments, the antibody binds one or two epitopeschosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO: 149), and HKPGGG(SEQ ID NO: 150). In some embodiments, the antibody binds all threeepitopes.

Further contemplated is an anti-tau antibody, or a tau-binding fragmentthereof, wherein said antibody or binding fragment comprises thefollowing complete chains:

a heavy chain having the amino acid sequence of SEQ ID NO. 32; and

a light chain variable domain having the amino acid sequence of any oneof SEQ ID NO. 57. In some embodiments, the antibody binds one or twoepitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO: 149),and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibody binds allthree epitopes.

Further contemplated is an anti-tau antibody, or a tau-binding fragmentthereof, wherein said antibody or binding fragment comprises thefollowing complete chains:

a heavy chain having the amino acid sequence of SEQ ID NO. 31; and

a light chain variable domain having the amino acid sequence of SEQ IDNO. 58. In some embodiments, the antibody binds one or two epitopeschosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO: 149), and HKPGGG(SEQ ID NO: 150). In some embodiments, the antibody binds all threeepitopes.

Further contemplated is an anti-tau antibody, or a tau-binding fragmentthereof, wherein said antibody or binding fragment comprises thefollowing complete chains:

a heavy chain having the amino acid sequence of SEQ ID NO. 32; and

a light chain variable domain having the amino acid sequence of SEQ IDNO. 58 and 27. In some embodiments, the antibody binds one or twoepitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO: 149),and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibody binds allthree epitopes.

Further contemplated is an anti-tau antibody, or a tau-binding fragmentthereof, wherein said antibody or binding fragment comprises thefollowing complete chains:

a heavy chain having the amino acid sequence of SEQ ID NO. 46; and

a light chain variable domain having the amino acid sequence of SEQ IDNO. 57. In some embodiments, the antibody binds one or two epitopeschosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO: 149), and HKPGGG(SEQ ID NO: 150). In some embodiments, the antibody binds all threeepitopes.

Further contemplated is an anti-tau antibody, or a tau-binding fragmentthereof, wherein said antibody or binding fragment comprises thefollowing complete chains:

a heavy chain having the amino acid sequence of SEQ ID NO. 47; and

a light chain variable domain having the amino acid sequence of SEQ IDNO. 57. In some embodiments, the antibody binds one or two epitopeschosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO: 149), and HKPGGG(SEQ ID NO: 150). In some embodiments, the antibody binds all threeepitopes.

Further contemplated is an anti-tau antibody, or a tau-binding fragmentthereof, wherein said antibody or binding fragment comprises thefollowing complete chains:

a heavy chain having the amino acid sequence of SEQ ID NO. 46; and

a light chain variable domain having the amino acid sequence of SEQ IDNO. 58. In some embodiments, the antibody binds one or two epitopeschosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO: 149), and HKPGGG(SEQ ID NO: 150). In some embodiments, the antibody binds all threeepitopes.

Further contemplated is an anti-tau antibody, or a tau-binding fragmentthereof, wherein said antibody or binding fragment comprises thefollowing complete chains:

a heavy chain having the amino acid sequence of SEQ ID NO. 47; and

a light chain variable domain having the amino acid sequence of SEQ IDNO. 58. In some embodiments, the antibody binds one or two epitopeschosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO: 149), and HKPGGG(SEQ ID NO: 150). In some embodiments, the antibody binds all threeepitopes.

It is also contemplated an antibody comprising:

a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 ofSEQ ID NOs 1, 2, 3, and being at least 85% identical to any one of SEQID NO. RHA, SEQ ID RHB, SEQ ID RHC, SEQ ID RHD, SEQ ID RHE, SEQ ID RHF,SEQ ID RHG, SEQ ID RHH, SEQ ID RHI, SEQ ID RHJ, SEQ ID RHL, SEQ ID RHM,i.e., SEQ ID NOs. 13-25;

and a mature light chain variable region comprising CDR-L1, CDR-L2, andCDR-L3 of SEQ ID NOs 4, 5, 6, respectively, and being at least 85%identical to SEQ ID NO. 26, RKA, wherein the antibody binds one or twoepitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO: 149),and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibody binds oneor two epitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO:149), and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibodybinds all three epitopes. In some embodiments, the antibody is chimeric.In some embodiments, the antibody is humanized.

It is also contemplated an antibody comprising:

a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 ofSEQ ID NOs 1, 2, 3, and being at least 90% identical to any one of SEQID NO:RHA, SEQ ID RHB, SEQ ID RHC, SEQ ID RHD, SEQ ID RHE, SEQ ID RHF,SEQ ID RHG, SEQ ID RHH, SEQ ID RHI, SEQ ID RHJ, SEQ ID RHL, SEQ ID RHM,i.e., SEQ ID NOs. 13-25;

and a mature light chain variable region comprising CDR-L1, CDR-L2, andCDR-L3 of SEQ ID NOs 4, 5, 6, respectively, and being at least 90%identical to SEQ ID NO: 26, RKA, wherein the antibody binds one or twoepitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO: 149),and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibody binds oneor two epitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO:149), and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibodybinds all three epitopes. In some embodiments, the antibody is chimeric.In some embodiments, the antibody is humanized.

It is also contemplated an antibody comprising:

a heavy chain variable region comprising CDR-H1; CDR-H2, and CDR-H3 ofSEQ ID NOs 1, 2, 3, and being at least 95% identical to any one of SEQID NO:RHA, SEQ ID RHB, SEQ ID RHC, SEQ ID RHD, SEQ ID RHE, SEQ ID RHF,SEQ ID RHG, SEQ ID RHH, SEQ ID RHI, SEQ ID RHJ, SEQ ID RHL, SEQ ID RHM,i.e., SEQ ID NOs. 13-25;

and a mature light chain variable region comprising CDR-L1, CDR-L2, andCDR-L3 of SEQ ID NOs 4, 5, 6, respectively, and being at least 95%identical to SEQ ID NO: 26, RKA, wherein the antibody binds one or twoepitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO: 149),and HKPGGG (SEQ ID NO: 150).

In some embodiments, the antibody binds one or two epitopes chosen fromHQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO: 149), and HKPGGG (SEQ ID NO:150). In some embodiments, the antibody binds all three epitopes. Insome embodiments, the antibody is chimeric. In some embodiments, theantibody is humanized.

Such antibody and binding fragment, according to the three previousparagraphs, is also contemplated in a form, wherein heavy chain position9 is occupied by P, position 21 is occupied by P, position 27 isoccupied by Y, position 28 is occupied by I, position 30 is occupied byT, position 38 is occupied by K, position 48 is occupied by I, position67 is occupied by K, position 68 is occupied by A, position 70 isoccupied by L, and/or position 95 is occupied by F. In one embodiment,the fight chain position 5 is occupied by S. In some embodiments, onlytwo of these 11 positions are occupied as such. In some embodiments,only three of these 11 positions are occupied as such. In someembodiments, only four of these 11 positions are occupied as such. Insome embodiments, only five of these 11 positions are occupied as such.In some embodiments, only six of these 11 positions are occupied assuch. In some embodiments, only seven of these 11 positions are occupiedas such. In some embodiments, only eight of these 11 positions areoccupied as such. In some embodiments, only nine of these 11 positionsare occupied as such. In some embodiments, only ten of these 11positions are occupied as such. In some embodiments, all 11 of these 11positions are occupied as such. In some embodiments, the antibody bindsone or two epitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ IDNO: 149), and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibodybinds all three epitopes.

And also contemplated is an antibody comprising:

a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 ofSEQ ID NOs. 1, 2, 3, and being at least 85% identical to any one of SEQID NO:RHA, SEQ ID RHB, SEQ ID RHC, SEQ ID RHD, SEQ ID RHE, SEQ ID RHF,SEQ ID RHG, SEQ ID RHH, SEQ ID RHI, SEQ ID RHJ, SEQ ID RHL, SEQ ID RHM,i.e., SEQ ID NOs. 13-25;

and a mature light chain variable region comprising CDR-L1, CDR-L2, andCDR-L3 of SEQ ID NOs 4, 5, 6, respectively, and being at least 85%identical to SEQ ID NO: 27 RKB, wherein the antibody antibody binds oneor two epitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO:149), and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibodybinds one or two epitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG(SEQ ID NO: 149), and HKPGGG (SEQ ID NO: 150). In some embodiments, theantibody binds all three epitopes. In some embodiments, the antibody ischimeric. In some embodiments, the antibody is humanized.

And also contemplated is an antibody comprising:

a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 ofSEQ ID NOs. 1, 2, 3, and being at least 90% identical to any one of SEQID NO:RHA, SEQ ID RHB, SEQ ID RHC, SEQ ID RHD, SEQ ID RHE, SEQ ID RHF,SEQ ID RHG, SEQ ID RHH, SEQ ID RHI, SEQ ID RHJ, SEQ ID RHL, SEQ ID RHM,i.e., SEQ ID NOs. 13-25;

and a mature light chain variable region comprising CDR-L1, CDR-L2, andCDR-L3 of SEQ ID NOs 4, 5, 6, respectively, and being at least 90%identical to SEQ ID NO: 27 RKB, wherein the antibody antibody binds oneor two epitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO:149), and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibodybinds one or two epitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG(SEQ ID NO: 149), and HKPGGG (SEQ ID NO: 150). In some embodiments, theantibody binds all three epitopes. In some embodiments, the antibody ischimeric. In some embodiments, the antibody is humanized.

And also contemplated is an antibody comprising:

a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 ofSEQ ID NOs. 1, 2, 3, and being at least 95% identical to any one of SEQID NO:RHA, SEQ ID RHB, SEQ ID RHC, SEQ ID RHD, SEQ ID RHE, SEQ ID RHF,SEQ ID RHG, SEQ ID RHH, SEQ ID RHI, SEQ ID RHJ, SEQ ID RHL, SEQ ID RHM,i.e., SEQ ID NOs. 13-25;

and a mature light chain variable region comprising CDR-L1, CDR-L2, andCDR-L3 of SEQ ID NOs 4, 5, 6, respectively, and being at least 95%identical to SEQ ID NO: 27 RKB, wherein the antibody antibody binds oneor two epitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO:149), and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibodybinds one or two epitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG(SEQ ID NO: 149), and HKPGGG (SEQ ID NO: 150). In some embodiments, theantibody binds all three epitopes. In some embodiments, the antibody ischimeric. In some embodiments, the antibody is humanized.

Such antibody and binding fragment, according to the three previousparagraphs, is also contemplated in a form, wherein heavy chain position9 is occupied by P, position 21 is occupied by P, position 27 isoccupied by Y, position 28 is occupied by I, position 30 is occupied byT, position 38 is occupied by K, position 48 is occupied by I, position67 is occupied by K, position 68 is occupied by A, position 70 isoccupied by L, and/or position 95 is occupied by F. In one embodiment,the light chain position 5 is occupied by S. In some embodiments, onlytwo of these 11 positions are occupied as such. In some embodiments,only three of these 11 positions are occupied as such. In someembodiments, only four of these 11 positions are occupied as such. Insome embodiments, only five of these 11 positions are occupied as such.In some embodiments, only six of these 11 positions are occupied assuch. In some embodiments, only seven of these 11 positions are occupiedas such. In some embodiments, only eight of these 11 positions areoccupied as such. In some embodiments, only nine of these 11 positionsare occupied as such. In some embodiments, only ten of these 11positions are occupied as such. In some embodiments, all 11 of these 11positions are occupied as such. In some embodiments, the antibody bindsone or two epitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ IDNO: 149), and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibodybinds all three epitopes.

The present disclosure also provides or contemplates an antibody in allthe previous paragraphs of this section (Summary of the Description),wherein said antibody or binding fragment is a Fab, Fab′, F(ab′)2, Fd,scFv, (scFv)₂, or scFv-Fc. In some embodiments, such an antibody bindsone or two epitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ IDNO: 149), and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibodybinds all three epitopes.

The present disclosure also provides or contemplates an antibody in theprevious paragraphs of this section, wherein said antibody or bindingfragment is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments,the antibody binds one or two epitopes chosen from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO: 149), and HKPGGG (SEQ ID NO: 150). In someembodiments, the antibody binds all three epitopes.

The present disclosure also provides or contemplates an antibody in theprevious paragraphs of this section, wherein said antibody or bindingfragment is an IgG1 antibody. In some embodiments, the antibody bindsone or two epitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ IDNO: 149), and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibodybinds all three epitopes.

The present disclosure also provides or contemplates an antibody in theprevious paragraphs of this section, wherein said antibody or bindingfragment is glycosylated. In some embodiments, the antibody binds one ortwo epitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO:149), and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibodybinds all three epitopes.

The present disclosure also provides or contemplates an antibody in theprevious paragraphs of this section, wherein said antibody or bindingfragment binds to Tau 151-391/4R with an affinity (K_(D)) of at least5×10⁻⁷. In some embodiments, the antibody binds one or two epitopeschosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO: 149), and HKPGGG(SEQ ID NO: 150). In some embodiments, the antibody binds all threeepitopes. In some embodiments, the antibody is chimeric. In someembodiments, the antibody is humanized. In some embodiments, the bindingaffinity is measured by SPR. In some embodiments, the binding affinityis measured by ELISA.

The present disclosure also provides or contemplates an antibody as inthe previous paragraphs of this section, wherein said antibody orbinding fragment binds to tau with at least 80% of the same bindingaffinity, substantially the same binding affinity, or better bindingaffinity, than the DC8E8 antibody secreted by hybridoma PTA-11994,deposited at the American Type Culture Collection. In some embodiments,the antibody binds one or two epitopes chosen from HQPGGG (SEQ ID NO:148), HVPGGG (SEQ ID NO: 149), and HKPGGG (SEQ ID NO: 150). In someembodiments, the antibody binds all three epitopes. In some embodiments,the antibody is chimeric. In some embodiments, the antibody ishumanized. In some embodiments, the binding affinity is measured by SPR.In some embodiments, the binding affinity is measured by ELISA.

The present disclosure also provides or contemplates an antibody as inthe previous paragraphs of this section, wherein said antibody competesfor binding to tau, at least one of the same epitope(s), with the DC8E8antibody secreted by hybridoma PTA-11994, deposited at the American TypeCulture Collection. In some embodiments, the antibody binds one or twoepitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO: 149),and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibody binds allthree epitopes. In some embodiments, the antibody is chimeric. In someembodiments, the antibody is humanized.

The present disclosure also provides or contemplates an antibody as inthe previous paragraphs of this section, wherein said antibody isrecombinantly produced. In some embodiments, the antibody binds one ortwo epitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO:149), and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibodybinds all three epitopes. In some embodiments, the antibody is chimeric.In some embodiments, the antibody is humanized.

In some embodiments, the antibody and binding fragment of the previousfour paragraphs are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 14, RHB, and the sequence of the lightchain variable region is SEQ ID NO. 26, RKA. Optionally, the antibodyand binding fragment are of the IgG1 isotype.

In some other embodiments, the antibody and binding fragment of thefirst four of the previous five paragraphs are also such that thesequence of the heavy chain variable region is SEQ ID NO. 16, RHD, andthe sequence of the light chain variable region is SEQ ID NO. 26, RKA.Optionally, the antibody and binding fragment are of the IgG1 isotype.

In some other embodiments, the antibody and binding fragment of thefirst four of the previous six paragraphs are also such that thesequence of the heavy chain variable region is SEQ ID NO. 17, RHE, andthe sequence of the light chain variable region is SEQ ID NO. 26, RKA.Optionally, the antibody and binding fragment are of the IgG1 isotype.

In some other embodiments, the antibody and binding fragment of thefirst four of the previous seven paragraphs are also such that thesequence of the heavy chain variable region is SEQ ID NO. 25, RHM, andthe sequence of the light chain variable region is SEQ ID NO. 26, RKA.Optionally, the antibody and binding fragment are of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14RHB and the sequence of the light chain variable region is SEQ ID NO. 26RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.16 RHD and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.17 RHE and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.25 RHM and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In some embodiments, the antibody and binding fragment of the first fourof the previous eight paragraphs are also such that the sequence of theheavy chain variable region is SEQ ID NO. 26, RHD, the sequence of thelight chain variable region is SEQ ID NO. 27, RKB, and the constantregion is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment of the first fourof the previous nine paragraphs are also such that the sequence of theheavy chain variable region is SEQ ID NO. 17, RHE, the sequence of thelight chain variable region is SEQ ID NO. 27, RKB, and the constantregion is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment of the first fourof the previous ten paragraphs are also such that the sequence of theheavy chain variable region is SEQ ID NO. 16, RHD, the sequence of thelight chain variable region is SEQ ID NO. 27, RKB, and the constantregion is of the IgG4 isotype.

In some embodiments, the antibody and binding fragment of the first fourof the previous eleven paragraphs are also such that the sequence of theheavy chain variable region is SEQ ID NO. 17, RHE, the sequence of thelight chain variable region is SEQ ID NO. 27, RKB, and the constantregion is of the IgG4 isotype.

The present disclosure also provides or contemplates an antibody as inthe previous paragraphs of this section, wherein said antibody isrecombinantly produced in a Chinese Hamster Ovary (CHO) cell line. Insome embodiments, the antibody binds one or two epitopes chosen fromHQPGGG (SEQ ID NO: 148), HVPGGG (SEQ NO: 149), and HKPGGG (SEQ ID NO:150). In some embodiments, the antibody binds all three epitopes.

The present disclosure also provides or contemplates an antibody as inthe previous paragraphs of this section, wherein said antibody orbinding fragment contains an Fc region that has been modified to altereffector function, half-life, proteolysis, and/or glycosylation. In someembodiments, the antibody binds one or two epitopes chosen from HQPGGG(SEQ ID NO: 148), HVPGGG (SEQ ID NO: 149), and HKPGGG (SEQ ID NO: 150).In some embodiments, the antibody binds all three epitopes.

The following are fourteen exemplary additional embodiments of theprevious two paragraphs:

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14RHB and the sequence of the light chain variable region is SEQ ID NO. 26RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.16 RHD and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.17 RHE and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.25 RHM and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 26,RHD, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 16,RHD, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14RHB and the sequence of the light chain variable region is SEQ ID NO. 26RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.16 RHD and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.17 RHE and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.25 RHM and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

The present disclosure also provides or contemplates an antibody in theprevious paragraphs of this section, wherein said antibody or bindingfragment is modified to modulate a functional characteristic selectedfrom the group consisting of antibody-dependent cellular cytotoxicity,complement-dependent cytotoxicity, serum half-life, biodistribution, andbinding to Fc receptors. In some embodiments, the antibody binds one ortwo epitopes chosen from HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO:149), and HKPGGG (SEQ ID NO: 150). In some embodiments, the antibodybinds all three epitopes.

The following are fourteen exemplary additional embodiments of theprevious paragraph:

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14RHB and the sequence of the light chain variable region is SEQ ID NO. 26RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.16 RHD and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.17 RHE and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.25 RHM and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 26,RHD, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 16,RHD, the sequence of the light chain variable region is SEQ ID NO. 27,RKB and the constant region is of the IgG4 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, the sequence of the light chain variable region is SEQ ID NO. 27,RKB and the constant region is of the IgG4 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14RHB and the sequence of the light chain variable region is SEQ ID NO. 26RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.16 RHD and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.17 RHE and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.25 RHM and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

The present disclosure also provides or contemplates an antibody as inthe previous paragraphs of this section, wherein said antibody has athermostability temperature equal to or greater than 69° C.

The following are fourteen exemplary additional embodiments of theprevious paragraph:

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14RHB and the sequence of the light chain variable region is SEQ ID NO. 26RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.16 RHD and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.17 RHE and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.25 RHM and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 26,RHD, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 16,RHD, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14RHB and the sequence of the light chain variable region is SEQ ID NO. 26RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.16 RHD and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.17 RHE and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.25 RHM and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In another aspect of the present disclosure, it contemplates acomposition comprising one of the antibodies and/or binding fragments ofthe present disclosure and another component.

In another aspect of the present disclosure, it contemplates apharmaceutical composition comprising the antibody or binding fragmentas described in this section and a pharmaceutically acceptable carrier,diluent, excipient, or stabilizer.

The following are fourteen exemplary additional embodiments of theprevious two paragraphs:

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14RHB and the sequence of the light chain variable region is SEQ ID NO. 26RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.16 RHD and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.17 RHE and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.25 RHM and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 26,RHD, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 16,RHD, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14RHB and the sequence of the light chain variable region is SEQ ID NO. 26RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.16 RHD and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.17 RHE and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.25 RHM and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

Also contemplated is a further version of any of the previouscomposition, wherein the composition comprises a lyophilized powder ofthe antibody or binding fragment.

Also contemplated are any of the previous compositions, wherein thecomposition is formulated for infusion or subcutaneous administration.

Also contemplated are any of these compositions, further comprising asecond therapeutic agent (also called combinations).

The following are fourteen exemplary additional composition embodimentsof the previous three paragraphs:

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14RHB and the sequence of the light chain variable region is SEQ ID NO. 26RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.16 RHD and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.17 RHE and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.25 RHM and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 26.RHD, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 16,RHD, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14RHB and the sequence of the light chain variable region is SEQ ID NO. 26RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.16 RHD and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.17 RHE and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.25 RHM and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In some of these compositions, the therapeutic agent and the antibody orbinding fragment are chemically conjugated.

In some of these compositions, the second therapeutic agent is useful inthe prophylaxis and/or treatment of AD.

It is also contemplated that the second therapeutic agent be selectedfrom, for example, beta-amyloid peptides (e.g., N-terminal amyloid betapeptides), which might or might not be conjugated to other compounds,such as mutated diphtheria toxin; other anti-tau antibodies, antibodiesagainst beta-amyloid, such as bapineuzumab, solaneuzumab, gantenerumab,crenezumab, and IVIG immunoglobulin, other immunization therapiestargeting Abeta oligomers, compounds preventing the hyperphosphorylationof tau, compounds preventing tau oligomerization and aggregation ordepolymerize tau oligomers (e.g. methylthioninium, rember or LMTX) andother active and passive immunization therapies targeting tauaggregates; and any pharmaceutically acceptable salts thereof.

It is also contemplated that the second therapeutic agent be selectedfrom amyloid-beta aggregation inhibitors (e.g., Tramiprosate),gamma-secretase inhibitors (e.g., semagacestat), and gamma-secretasemodulators (tarenflurbil); and any pharmaceutically acceptable saltsthereof.

The following are fourteen exemplary additional embodiments of theprevious four paragraphs:

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14RHB and the sequence of the light chain variable region is SEQ ID NO. 26RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.16 RHD and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.17 RHE and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.25 RHM and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14RHB and the sequence of the light chain variable region is SEQ ID NO. 26RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.16 RHD and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.17 RHE and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.25 RHM and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 26,RHD, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 16,RHD, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In some instances, the second therapeutic agent is selected fromacetylcholinesterase inhibitors (e.g., donepezil, rivastigmine,galantamine, tacrine, nutritive supplements), N-Methyl-D-aspartate(NMDA) receptor antagonists (e.g., memantine), inhibitors of DNA repair(e.g., pirenzepine or a metabolite thereof), transition metal chelators,growth factors, hormones, non-steroidal anti-inflammatory drugs (NSAID),antioxidants, lipid lowering agents, selective phosphodiesteraseinhibitors, inhibitors of tau aggregation, inhibitors of proteinkinases, inhibitors of anti-mitochondrial dysfunction drugs,neurotrophins, inhibitors of heat shock proteins, inhibitors ofLipoprotein-associated phospholipase A₂, memantine, an anti-apoptoticcompound, a metal chelator, an inhibitor of DNA repair,3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate (1,3PDS),a secretase activator, a beta-secretase inhibitor, a gamma-secretaseinhibitor, a beta-amyloid peptide, a beta-amyloid antibody, a taupeptide, a neurotransmitter, a beta-sheet breaker, an anti-inflammatorymolecule: and any pharmaceutically acceptable salts thereof.

The following are fourteen exemplary additional embodiments of theprevious paragraph:

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14RHB and the sequence of the light chain variable region is SEQ ID NO. 26RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.16 RHD and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.17 RHE and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.25 RHM and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14RHB and the sequence of the light chain variable region is SEQ ID NO. 26RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.16 RHD and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.17 RHE and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.25 RHM and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 16,RHD, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 16,RHD, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

Also contemplated are compositions wherein the second therapeutic agentis selected from compounds described in WO 2004/058258 (see especiallypages 16 and 17) including therapeutic drug targets (page 36-39),alkanesulfonic acids and alkanolsulfuric acids (pages 39-51),cholinesterase inhibitors (pages 51-56), NMDA receptor antagonists(pages 56-58), estrogens (pages 58-59), non-steroidal anti-inflammatorydrugs (pages 60-61), antioxidants (pages 61-62), peroxisomeproliferators-activated receptor (PPAR) agonists (pages 63-67),cholesterol-lowering agents (pages 68-75); amyloid inhibitors (pages75-77), amyloid formation inhibitors (pages 77-78), metal chelators(pages 78-79), anti-psychotics and anti-depressants (pages 80-82),nutritional supplements (pages 83-89) and compounds increasing theavailability of biologically active substances in the brain (see pages89-93) and prodrugs (pages 93 and 94); and any pharmaceuticallyacceptable salts thereof.

Also contemplated are compositions wherein the second therapeutic agentis selected from compounds preventing tau oligomerization andaggregation and compounds that depolymerize tau oligomers (e.g.methylthioninium, rember or LMTX).

The following are fourteen exemplary additional embodiments of theprevious two paragraphs:

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14RHB and the sequence of the light chain variable region is SEQ ID NO. 26RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.16 RHD and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.17 RHE and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.25 RHM and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG1isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 26,RHD, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 16,RHD, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 17,RHE, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14RHB and the sequence of the light chain variable region is SEQ ID NO. 26RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.16 RHD and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.17 RHE and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some other embodiments, the antibody and binding fragment are alsosuch that the sequence of the heavy chain variable region is SEQ ID NO.25 RHM and the sequence of the light chain variable region is SEQ ID NO.26 RKA. Optionally, the antibody and binding fragment are of the IgG4isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG1 isotype.

In some embodiments, the antibody and binding fragment are also suchthat the sequence of the heavy chain variable region is SEQ ID NO. 14,RHB, the sequence of the light chain variable region is SEQ ID NO. 27,RKB, and the constant region is of the IgG4 isotype.

In another aspect, the disclosure provides a diagnostic reagentcomprising the antibody or binding fragment according to any one ofclaims 1-51 and a carrier, diluent, excipient, or stabilizer.

In some embodiments of these diagnostic reagents, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. RHB and the sequence of the light chainvariable region is SEQ ID NO. RKA. Optionally, the antibody and bindingfragment are of the IgG1 isotype.

In some other embodiments of these diagnostic reagents, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 16 RHD and the sequence of the light chainvariable region is SEQ ID NO. 26 RKA. Optionally, the antibody andbinding fragment are of the IgG1 isotype.

In some other embodiments of these diagnostic reagents, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 17 RHE and the sequence of the light chainvariable region is SEQ ID NO. 26 RKA. Optionally, the antibody andbinding fragment are of the IgG1 isotype.

In some other embodiments of these diagnostic reagents, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 25 RHM and the sequence of the light chainvariable region is SEQ ID NO. 26 RKA. Optionally, the antibody andbinding fragment are of the IgG1 isotype.

In some embodiments of these diagnostic reagents, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 26, RHD, the sequence of the light chainvariable region is SEQ ID NO. 27, RKB, and the constant region is of theIgG1 isotype.

In some embodiments of these diagnostic reagents, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 17, RHE, the sequence of the light chainvariable region is SEQ ID NO. 27, RKB, and the constant region is of theIgG1 isotype.

In some embodiments of these diagnostic reagents, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 16, RHD, the sequence of the light chainvariable region is SEQ ID NO. 27, RKB, and the constant region is of theIgG4 isotype.

In some embodiments of these diagnostic reagents, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 17, RHE, the sequence of the light chainvariable region is SEQ ID NO. 27, RKB, and the constant region is of theIgG4 isotype.

In some embodiments of these diagnostic reagents, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 14 RHB and the sequence of the light chainvariable region is SEQ ID NO. 26 RKA. Optionally, the antibody andbinding fragment are of the IgG4 isotype.

In some other embodiments of these diagnostic reagents, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 16 RHD and the sequence of the light chainvariable region is SEQ ID NO. 26 RKA. Optionally, the antibody andbinding fragment are of the IgG4 isotype.

In some other embodiments of these diagnostic reagents, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 17 RHE and the sequence of the light chainvariable region is SEQ ID NO. 26 RKA. Optionally, the antibody andbinding fragment are of the IgG4 isotype.

In some other embodiments of these diagnostic reagents, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 25 RHM and the sequence of the light chainvariable region is SEQ ID NO. 26 RKA. Optionally, the antibody andbinding fragment are of the IgG4 isotype.

In some embodiments of these diagnostic reagents, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 14, RHB, the sequence of the light chainvariable region is SEQ ID NO. 27, RKB, and the constant region is of theIgG1 isotype.

In some embodiments of these diagnostic reagents, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 14, RHB, the sequence of the light chainvariable region is SEQ ID NO. 27, RKB, and the constant region is of theIgG4 isotype.

In yet another aspect, the disclosure provides an immunoconjugate havingthe formula (A)-(L)-(C), wherein: (A) is an antibody or binding fragmentthereof of any one of claims 1-51; (L) is a linker; and (C) is an agent;and wherein said linker (L) links (A) to (C). In some cases, (C) is atherapeutic agent, an imaging agent, a detectable agent, or a diagnosticagent. In some cases, (C) is a therapeutic agent.

In some embodiments of these immunoconjugates, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 14 RHB and the sequence of the light chain variableregion is SEQ ID NO. 26 RKA. Optionally, the antibody and bindingfragment are of the IgG1 isotype.

In some other embodiments of these immunoconjugates, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 16 RHD and the sequence of the light chainvariable region is SEQ ID NO. 26 RKA. Optionally, the antibody andbinding fragment are of the IgG1 isotype.

In some other embodiments of these immunoconjugates, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 17 RHE and the sequence of the light chainvariable region is SEQ ID NO. 26 RKA. Optionally, the antibody andbinding fragment are of the IgG1 isotype.

In some other embodiments of these immunoconjugates, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 25 RHM and the sequence of the light chainvariable region is SEQ ID NO. 26 RKA. Optionally, the antibody andbinding fragment are of the IgG1 isotype.

In some embodiments of these immunoconjugates, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 26, RHD, the sequence of the light chain variableregion is SEQ ID NO. 27, RKB, and the constant region is of the IgG1isotype.

In some embodiments of these immunoconjugates, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHE, the sequence of the light chain variableregion is SEQ ID NO. 27, RKB, and the constant region is of the IgG1isotype.

In some embodiments of these immunoconjugates, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD, the sequence of the light chain variableregion is SEQ ID NO. 27, RKB, and the constant region is of the IgG4isotype.

In some embodiments of these immunoconjugates, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHE, the sequence of the light chain variableregion is SEQ ID NO. 27, RKB, and the constant region is of the IgG4isotype.

In some embodiments of these immunoconjugates, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 14 RHB and the sequence of the light chain variableregion is SEQ ID NO. 26 RKA. Optionally, the antibody and bindingfragment are of the IgG4 isotype.

In some other embodiments of these immunoconjugates, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 16 RHD and the sequence of the light chainvariable region is SEQ ID NO. 26 RKA. Optionally, the antibody andbinding fragment are of the IgG4 isotype.

In some other embodiments of these immunoconjugates, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 17 RHE and the sequence of the light chainvariable region is SEQ ID NO. 26 RKA. Optionally, the antibody andbinding fragment are of the IgG4 isotype.

In some other embodiments of these immunoconjugates, the antibody andbinding fragment are also such that the sequence of the heavy chainvariable region is SEQ ID NO. 25 RHM and the sequence of the light chainvariable region is SEQ ID NO. 26 RKA. Optionally, the antibody andbinding fragment are of the IgG4 isotype.

In some embodiments of these immunoconjugates, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 14, RHB, the sequence of the light chain variableregion is SEQ ID NO. 27, RKB, and the constant region is of the IgG1isotype.

In some embodiments of these immunoconjugates, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 14, RHB, the sequence of the light chain variableregion is SEQ ID NO. 27, RKB, and the constant region is of the IgG4isotype.

The invention contemplates nucleic acid molecules (RNA or DNA) encodingany of the antibodies and tau-binding fragments disclosed above. In oneembodiment, such nucleic acid molecules comprise one or more of nucleicacid sequences chosen from SEQ ID NOs. 96-124, 141-142, and 127-140. Inother embodiments, such nucleic acid molecules comprise one or more ofnucleic acid sequences chosen from sequences that are at least 85%identical to any one of SEQ ID NOs. 96-124, 141-142, and 127-140. Inother embodiments, such nucleic acid molecules comprise one or more ofnucleic acid sequences chosen from sequences that are at least 90%identical to any one of SEQ ID NOs. 96-124, 141-142, and 127-140. Inother embodiments, such nucleic acid molecules comprise one or more ofnucleic acid sequences chosen from sequences that are at least 95%identical to any one of SEQ ID NOs. 96-124, 141-142, and 127-140, Inother embodiments, such nucleic acid molecules comprise one or more ofnucleic acid sequences chosen from sequences that are at least 96%identical to any one of SEQ ID NOs. 96-124, 141-142, and 127-140. Inother embodiments, such nucleic acid molecules comprise one or more ofnucleic acid sequences chosen from sequences that are at least 97%identical to any one of SEQ ID NOs. 96-124, 141-142, and 127-140. Inother embodiments, such nucleic acid molecules comprise one or more ofnucleic acid sequences chosen from sequences that are at least 98%identical to any one of SEQ ID NOs. 96-124, 141-142, and 127-140. Inother embodiments, such nucleic acid molecules comprise one or more ofnucleic acid sequences chosen from sequences that are at least 99%identical to any one of SEQ ID NOs. 96-124, 141-142, and 127-140.

In one embodiment, the disclosure contemplates a nucleic acid moleculecomprising a nucleotide sequence encoding a heavy chain variable region(HCVR) or a tau-binding fragment thereof of an anti-tau antibody,wherein said HCVR or fragment thereof comprises: (i) a framework derivedfrom human immunoglobulin M65092 (SEQ ID NO. 71), (ii) an HCVR CDR1comprising the amino acid sequence of SEQ ID NO: 1, (iii) an HCVR CDR2comprising the amino acid sequence of SEQ ID NO: 2, and (iv) an HCVRCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and whereinsaid HCVR or fragment thereof comprises an amino acid sequence that isat least 98% identical to the amino acid sequence of anyone of SEQ IDNO: RHA through SEQ ID NO. RHM, i.e., SEQ ID NOs. 13-25. In each ofthese embodiments, it can also be the case that the antibody or fragmentthereof comprises a heavy chain further comprising an IgG1 constantregion. In other embodiments, the antibody or fragment thereof comprisesa heavy chain further comprising an IgG4 constant region.

A nucleic acid molecule comprising a nucleotide sequence encoding alight chain variable region (LCVR) or a tau-binding fragment thereof ofan anti-tau antibody, wherein said LCVR or fragment thereof comprises:(i) a framework derived from human immunoglobulin X72449 (SEQ ID NO.65), (ii) an LCVR CDR1 comprising the amino acid sequence of SEQ ID NO:4, (iii) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO:5, and (iv) an LCVR CDR3 comprising the amino acid sequence of SEQ IDNO: 6, and wherein said LCVR or fragment thereof comprises an amino acidsequence that is at least 98% identical to the amino acid sequence ofSEQ ID NO: 26, RKA, or SEQ ID NO. 27, RKB. In each of these embodiments,it can also be the case that the antibody or fragment thereof comprisesa heavy chain further comprising a kappa constant region.

In one embodiment, the disclosure contemplates a nucleic acid moleculecomprising a nucleotide sequence encoding a heavy chain variable region(HCVR) or a tau-binding fragment thereof of an anti-tau antibody,wherein said HCVR or fragment thereof comprises: (i) a framework derivedfrom human immunoglobulin M65092 (SEQ ID NO. 71), (ii) an HCVR CDR1comprising the amino acid sequence of SEQ ID NO: 1, (iii) an HCVR CDR2comprising the amino acid sequence of SEQ ID NO: 2, and (iv) an HCVRCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and whereinsaid HCVR or fragment thereof comprises an amino acid sequence that isidentical to the amino acid sequence of anyone of SEQ ID NO: RHA throughSEQ ID NO. RHM, i.e., SEQ ID NOs. 13-25. In each of these embodiments,it can also be the case that the antibody or fragment thereof comprisesa heavy chain further comprising an IgG1 constant region. In otherembodiments, the antibody or fragment thereof comprises a heavy chainfurther comprising an IgG4 constant region.

A nucleic acid molecule comprising a nucleotide sequence encoding alight chain variable region (LCVR) or a tau-binding fragment thereof ofan anti-tau antibody, wherein said LCVR or fragment thereof comprises:(i) a framework derived from human immunoglobulin X72449 (SEQ ID NO.65), (ii) an LCVR CDR1 comprising the amino acid sequence of SEQ ID NO:4, (iii) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO:5, and (iv) an LCVR CDR3 comprising the amino acid sequence of SEQ IDNO: 6, and wherein said LCVR or fragment thereof comprises an amino acidsequence that is identical to the amino acid sequence of SEQ ID NO: 26,RKA, or SEQ ID NO. 27, RKB. In each of these embodiments, it can also bethe case that the antibody or fragment thereof comprises a heavy chainfurther comprising a kappa constant region.

Also contemplated is a nucleic acid molecule comprising a nucleotidesequence encoding a heavy chain variable region (HCVR) or a tau-bindingfragment thereof of an anti-tau antibody, wherein said HCVR or fragmentthereof comprises: (i) a framework derived from human immunoglobulinM65092 (SEQ ID NO. 71), (ii) an HCVR CDR1 comprising the amino acidsequence of SEQ ID NO: 1, (iii) an HCVR CDR2 comprising the amino acidsequence of SEQ ID NO: 2, and (iv) an HCVR CDR3 comprising the aminoacid sequence of SEQ ID NO: 3, and wherein said HCVR or fragment thereofcomprises an amino acid sequence that is at least 98% identical to theamino acid sequence of SEQ ID NO: 14, RHB. In each of these embodiments,it can also be the case that the antibody or fragment thereof comprisesa heavy chain further comprising an IgG1 constant region.

Also contemplated is a nucleic acid molecule comprising a nucleotidesequence encoding a heavy chain variable region (HCVR) or a tau-bindingfragment thereof of an anti-tau antibody, wherein said HCVR or fragmentthereof comprises: (i) a framework derived from human immunoglobulinM65092 (SEQ ID NO. 71), (ii) an HCVR CDR1 comprising the amino acidsequence of SEQ ID NO: 1, (iii) an HCVR CDR2 comprising the amino acidsequence of SEQ ID NO: 2, and (iv) an HCVR CDR3 comprising the aminoacid sequence of SEQ ID NO: 3, and wherein said HCVR or fragment thereofcomprises an amino acid sequence that is at least 98% identical to theamino acid sequence of SEQ ID NO: 16, RHD. In each of these embodiments,it can also be the case that the antibody or fragment thereof comprisesa heavy chain further comprising an IgG1 constant region.

Also contemplated is a nucleic acid molecule comprising a nucleotidesequence encoding a heavy chain variable region (HCVR) or a tau-bindingfragment thereof of an anti-tau antibody, wherein said HCVR or fragmentthereof comprises: (i) a framework derived from human immunoglobulinM65092 (SEQ ID NO. 71), (ii) an HCVR CDR1 comprising the amino acidsequence of SEQ ID NO: 1, (iii) an HCVR CDR2 comprising the amino acidsequence of SEQ ID NO: 2, and (iv) an HCVR CDR3 comprising the aminoacid sequence of SEQ ID NO: 3, and wherein said HCVR or fragment thereofcomprises an amino acid sequence that is at least 98% identical to theamino acid sequence of SEQ ID NO: 17, RHE. In each of these embodiments,it can also be the case that the antibody or fragment thereof comprisesa heavy chain further comprising an IgG1 constant region.

Also contemplated is a nucleic acid molecule comprising a nucleotidesequence encoding a heavy chain variable region (HCVR) or a tau-bindingfragment thereof of an anti-tau antibody, wherein said HCVR or fragmentthereof comprises: (i) a framework derived from human immunoglobulinM65092 (SEQ ID NO. 71), (ii) an HCVR CDR1 comprising the amino acidsequence of SEQ ID NO: 1, (iii) an HCVR CDR2 comprising the amino acidsequence of SEQ ID NO: 2, and (iv) an HCVR CDR3 comprising the aminoacid sequence of SEQ ID NO: 3, and wherein said HCVR or fragment thereofcomprises an amino acid sequence that is at least 98% identical to theamino acid sequence of SEQ ID NO: 25, RHM. In each of these embodiments,it can also be the case that the antibody or fragment thereof comprisesa heavy chain further comprising an IgG1 constant region.

Also contemplated is a nucleic acid molecule comprising a nucleotidesequence encoding a heavy chain variable region (HCVR) or a tau-bindingfragment thereof of an anti-tau antibody, wherein said HCVR or fragmentthereof comprises: (i) a framework derived from human immunoglobulinM65092 (SEQ ID NO. 71), (ii) an HCVR CDR1 comprising the amino acidsequence of SEQ ID NO: 1, (iii) an HCVR CDR2 comprising the amino acidsequence of SEQ ID NO: 2, and (iv) an HCVR CDR3 comprising the aminoacid sequence of SEQ ID NO: 3, and wherein said HCVR or fragment thereofcomprises an amino acid sequence that is identical to the amino acidsequence of SEQ ID NO: 14, RHB. In each of these embodiments, it canalso be the case that the antibody or fragment thereof comprises a heavychain further comprising an IgG1 constant region.

Also contemplated is a nucleic acid molecule comprising a nucleotidesequence encoding a heavy chain variable region (HCVR) or a tau-bindingfragment thereof of an anti-tau antibody, wherein said HCVR or fragmentthereof comprises: (i) a framework derived from human immunoglobulinM65092 (SEQ ID NO. 71), (ii) an HCVR CDR1 comprising the amino acidsequence of SEQ ID NO: 1, (iii) an HCVR CDR2 comprising the amino acidsequence of SEQ ID NO: 2, and (iv) an HCVR CDR3 comprising the aminoacid sequence of SEQ ID NO: 3, and wherein said HCVR or fragment thereofcomprises an amino acid sequence that is identical to the amino acidsequence of SEQ ID NO: 16, RHD. In each of these embodiments, it canalso be the case that the antibody or fragment thereof comprises a heavychain further comprising an IgG1 constant region.

Also contemplated is a nucleic acid molecule comprising a nucleotidesequence encoding a heavy chain variable region (HCVR) or a tau-bindingfragment thereof of an anti-tau antibody, wherein said HCVR or fragmentthereof comprises: (i) a framework derived from human immunoglobulinM65092 (SEQ ID NO. 71), (ii) an HCVR CDR1 comprising the amino acidsequence of SEQ ID NO: 1, (iii) an HCVR CDR2 comprising the amino acidsequence of SEQ ID NO: 2, and (iv) an HCVR CDR3 comprising the aminoacid sequence of SEQ ID NO: 3, and wherein said HCVR or fragment thereofcomprises an amino acid sequence that is identical to the amino acidsequence of SEQ ID NO: 17, RHE. In each of these embodiments, it canalso be the case that the antibody or fragment thereof comprises a heavychain further comprising an IgG1 constant region.

Also contemplated is a nucleic acid molecule comprising a nucleotidesequence encoding a heavy chain variable region (HCVR) or a tau-bindingfragment thereof of an anti-tau antibody, wherein said HCVR or fragmentthereof comprises: (i) a framework derived from human immunoglobulinM65092 (SEQ ID NO. 71), (ii) an HCVR CDR1 comprising the amino acidsequence of SEQ ID NO: 1, (iii) an HCVR CDR2 comprising the amino acidsequence of SEQ ID NO: 2, and (iv) an HCVR CDR3 comprising the aminoacid sequence of SEQ ID NO: 3, and wherein said HCVR or fragment thereofcomprises an amino acid sequence that is identical to the amino acidsequence of SEQ ID NO: 25, RHM. In each of these embodiments, it canalso be the case that the antibody or fragment thereof comprises a heavychain further comprising an IgG1 constant region.

In some embodiments, any of the nucleic acid molecules just described inthis section is such that the antibody or fragment thereof comprises aheavy chain further comprising an IgG1 constant region.

Also contemplated is a nucleic acid molecule comprising a nucleotidesequence encoding a light chain variable region (LCVR) or a tau-bindingfragment thereof of an anti-tau antibody, wherein said LCVR or fragmentthereof comprises: (i) a framework derived from human immunoglobulinX72449 (SEQ ID NO. 65), (ii) an LCVR CDR1 comprising the amino acidsequence of SEQ ID NO: 4, (iii) an LCVR CDR2 comprising the amino acidsequence of SEQ ID NO: 5, and (iv) an LCVR CDR3 comprising the aminoacid sequence of SEQ ID NO: 6, and wherein said LCVR or fragment thereofcomprises an amino acid sequence that is at least 98% identical to theamino acid sequence of SEQ ID NO: 26, RKA. In each of these embodiments,it can also be the case that the antibody or fragment thereof comprisesa heavy chain further comprising a kappa constant region.

Also contemplated is a nucleic acid molecule comprising a nucleotidesequence encoding a light chain variable region (LCVR) or a tau-bindingfragment thereof of an anti-tau antibody, wherein said LCVR or fragmentthereof comprises: (i) a framework derived from human immunoglobulinX72449 (SEQ ID NO. 65), (ii) an LCVR CDR1 comprising the amino acidsequence of SEQ ID NO: 4, (iii) an LCVR CDR2 comprising the amino acidsequence of SEQ ID NO: 5, and (iv) an LCVR CDR3 comprising the aminoacid sequence of SEQ ID NO: 6, and wherein said LCVR or fragment thereofcomprises an amino acid sequence that is at least 98% identical to theamino acid sequence of SEQ ID NO: RKB. In each of these embodiments, itcan also be the case that the antibody or fragment thereof comprises aheavy chain further comprising a kappa constant region.

Also contemplated is a nucleic acid molecule comprising a nucleotidesequence encoding a light chain variable region (LCVR) or a tau-bindingfragment thereof of an anti-tau antibody, wherein said LCVR or fragmentthereof comprises: (i) a framework derived from human immunoglobulinX72449 (SEQ ID NO. 65), (ii) an LCVR CDR1 comprising the amino acidsequence of SEQ ID NO: 4, (iii) an LCVR CDR2 comprising the amino acidsequence of SEQ ID NO: 5, and (iv) an LCVR CDR3 comprising the aminoacid sequence of SEQ ID NO: 6, and wherein said LCVR or fragment thereofcomprises an amino acid sequence that is identical to the amino acidsequence of SEQ ID NO: 26, RKA. In each of these embodiments, it canalso be the case that the antibody or fragment thereof comprises a heavychain further comprising a kappa constant region.

Also contemplated is a nucleic acid molecule comprising a nucleotidesequence encoding a light chain variable region (LCVR) or a tau-bindingfragment thereof of an anti-tau antibody, wherein said LCVR or fragmentthereof comprises: (i) a framework derived from human immunoglobulinX72449 (SEQ ID NO. 65), (ii) an LCVR CDR1 comprising the amino acidsequence of SEQ ID NO: 4, (iii) an LCVR CDR2 comprising the amino acidsequence of SEQ ID NO: 5, and (iv) an LCVR CDR3 comprising the aminoacid sequence of SEQ ID NO: 6, and wherein said LCVR or fragment thereofcomprises an amino acid sequence that is identical to the amino acidsequence of SEQ ID NO: RKB. In each of these embodiments, it can also bethe case that the antibody or fragment thereof comprises a heavy chainfurther comprising a kappa constant region.

In some embodiments, the nucleic acid sequence encodes the HCVR of anyone of SEQ ID NO. 13-25 (RHA through RHM).

In yet another set of embodiments, the nucleic acid sequence encodes theHCVR of SEQ ID NO. 14, RHB.

In one embodiment of these nucleic acids, the nucleic acid sequenceencodes the HCVR of SEQ ID NO. 16, RHD

In one embodiment of these nucleic acids, the nucleic acid sequenceencodes the HCVR of SEQ ID NO. 17, RHE.

In one embodiment of these nucleic acids, the nucleic acid sequenceencodes the HCVR of SEQ ID NO. 25, RHM.

In one embodiment of these nucleic acids, the nucleic acid sequenceencodes the LCVR of any one of SEQ ID NO. 26, RKA, and 27, RKB.

Also contemplated is a nucleic acid sequence comprising a nucleotidesequence encoding a heavy chain variable region (HCVR) or a tau-bindingfragment thereof of an anti-tau antibody, wherein said HCVR or fragmentthereof comprises: (i) a framework derived from human immunoglobulinM65092 (SEQ ID NO.), (ii) an HCVR CDR1 comprising the amino acidsequence of SEQ ID NO: 1, (iii) an HCVR CDR2 comprising the amino acidsequence of SEQ ID NO: 2, and (iv) an HCVR CDR3 comprising the aminoacid sequence of SEQ ID NO: 3, and wherein said HCVR or fragment thereofcomprises an amino acid sequence that is at least 98% identical to theamino acid sequence of anyone of SEQ ID NO: RHA through SEQ ID NO. RHM(i.e., 13-25), and further such that the nucleic acid sequence comprisesa nucleotide sequence that hybridizes under stringent conditions to acomplementary strand of any one of SEQ ID NO: RHA through SEQ ID NO. RHM(i.e, 96-108), wherein the stringent hybridization conditions comprisehybridization in 5×SSPE, 1% SDS, 1×Denhardts solution at 65° C. andwashing in 2×SSC, 1% SDS and subsequently with 0.2×SSC at 65° C.

Also contemplated is a nucleic acid sequence comprising a nucleotidesequence encoding a heavy chain variable region (HCVR) or a tau-bindingfragment thereof of an anti-tau antibody, wherein said HCVR or fragmentthereof comprises: (i) a framework derived from human immunoglobulinM65092 (SEQ ID NO.), (ii) an HCVR CDR1 comprising the amino acidsequence of SEQ ID NO: 1, (iii) an HCVR CDR2 comprising the amino acidsequence of SEQ ID NO: 2, and (iv) an HCVR CDR3 comprising the aminoacid sequence of SEQ ID NO: 3, and wherein said HCVR or fragment thereofcomprises an amino acid sequence that is at least 98% identical to theamino acid sequence of SEQ ID NO. 14, RHB, and further such that thenucleic acid sequence comprises a nucleotide sequence that hybridizesunder stringent conditions to a complementary strand of SEQ ID NO. 97,RHB, wherein the stringent hybridization conditions comprisehybridization in 5×SSPE, 1% SDS, 1×Denhardts solution at 65° C. andwashing in 2×SSC, 1% SDS and subsequently with 0.2×SSC at 65° C.

Also contemplated is a nucleic acid sequence comprising a nucleotidesequence encoding a heavy chain variable region (HCVR) or a tau-bindingfragment thereof of an anti-tau antibody, wherein said HCVR or fragmentthereof comprises: (i) a framework derived from human immunoglobulinM65092 (SEQ ID NO.), (ii) an HCVR CDR1 comprising the amino acidsequence of SEQ ID NO: 1, (iii) an HCVR CDR2 comprising the amino acidsequence of SEQ ID NO: 2, and (iv) an HCVR CDR3 comprising the aminoacid sequence of SEQ ID NO: 3, and wherein said HCVR or fragment thereofcomprises an amino acid sequence that is at least 98% identical to theamino acid sequence of SEQ ID NO. 16, RHD, and further such that thenucleic acid sequence comprises a nucleotide sequence that hybridizesunder stringent conditions to a complementary strand of SEQ ID NO. 99,RHD, wherein the stringent hybridization conditions comprisehybridization in 5×SSPE, 1% SDS, 1×Denhardts solution at 65° C. andwashing in 2×SSC, 1% SDS and subsequently with 0.2×SSC at 65° C.

Also contemplated is a nucleic acid sequence comprising a nucleotidesequence encoding a heavy chain variable region (HCVR) or a tau-bindingfragment thereof of an anti-tau antibody, wherein said HCVR or fragmentthereof comprises: (i) a framework derived from human immunoglobulinM65092 (SEQ ID NO.), (ii) an HCVR CDR1 comprising the amino acidsequence of SEQ ID NO: 1, (iii) an HCVR CDR2 comprising the amino acidsequence of SEQ ID NO: 2, and (iv) an HCVR CDR3 comprising the aminoacid sequence of SEQ ID NO: 3, and wherein said HCVR or fragment thereofcomprises an amino acid sequence that is at least 98% identical to theamino acid sequence of SEQ ID NO. 17, RHE, and further such that thenucleic acid sequence comprises a nucleotide sequence that hybridizesunder stringent conditions to a complementary strand of SEQ ID NO. 100,RHE, wherein the stringent hybridization conditions comprisehybridization in 5×SSPE, 1% SDS, 1×Denhardts solution at 65° C. andwashing in 2×SSC, 1% SDS and subsequently with 0.2×SSC at 65° C.

Also contemplated is a nucleic acid sequence comprising a nucleotidesequence encoding a heavy chain variable region (HCVR) or a tau-bindingfragment thereof of an anti-tau antibody, wherein said HCVR or fragmentthereof comprises: (i) a framework derived from human immunoglobulinM65092 (SEQ ID NO.), (ii) an HCVR CDR1 comprising the amino acidsequence of SEQ ID NO: 1, (iii) an HCVR CDR2 comprising the amino acidsequence of SEQ ID NO: 2, and (iv) an HCVR CDR3 comprising the aminoacid sequence of SEQ ID NO: 3, and wherein said HCVR or fragment thereofcomprises an amino acid sequence that is at least 98% identical to theamino acid sequence of SEQ ID NO. 25, RHM, and further such that thenucleic acid sequence comprises a nucleotide sequence that hybridizesunder stringent conditions to a complementary strand of SEQ ID NO. 108,RHM, wherein the stringent hybridization conditions comprisehybridization in 5×SSPE, 1% SDS, 1×Denhardts solution at 65° C. andwashing in 2×SSC, 1% SDS and subsequently with 0.2×SSC at 65° C.

Also contemplated is a nucleic acid molecule comprising a nucleotidesequence encoding a light chain variable region (LCVR) or a tau-bindingfragment thereof of an anti-tau antibody, wherein said LCVR or fragmentthereof comprises: (i) a framework derived from human immunoglobulinX72449 (SEQ ID NO. 65), (ii) an LCVR CDR1 comprising the amino acidsequence of SEQ ID NO: 4, (iii) an LCVR CDR2 comprising the amino acidsequence of SEQ ID NO: 5, and (iv) an LCVR CDR3 comprising the aminoacid sequence of SEQ ID NO: 6, and wherein said LCVR or fragment thereofcomprises an amino acid sequence that is at least 98% identical to theamino acid sequence of SEQ ID NO: 26, RKA; and further such that thenucleic acid sequence comprises a nucleotide sequence that hybridizesunder stringent conditions to a complementary strand of SEQ ID NO: 109,RKA, wherein the stringent hybridization conditions comprisehybridization in 5×SSPE, 1% SDS, 1×Denhardts solution at 65° C. andwashing in 2×SSC, 1% SDS and subsequently with 0.2×SSC at 65° C.

Also contemplated is a nucleic acid molecule comprising a nucleotidesequence encoding a light chain variable region (LCVR) or a tau-bindingfragment thereof of an anti-tau antibody, wherein said LCVR or fragmentthereof comprises: (i) a framework derived from human immunoglobulinX72449 (SEQ ID NO. 65), (ii) an LCVR CDR1 comprising the amino acidsequence of SEQ ID NO: 4, (iii) an LCVR CDR2 comprising the amino acidsequence of SEQ ID NO: 5, and (iv) an LCVR CDR3 comprising the aminoacid sequence of SEQ ID NO: 6, and wherein said LCVR or fragment thereofcomprises an amino acid sequence that is at least 98% identical to theamino acid sequence of SEQ ID NO: 14, RKB; and further such that thenucleic acid sequence comprises a nucleotide sequence that hybridizesunder stringent conditions to a complementary strand of SEQ ID NO: 110,RKB, wherein the stringent hybridization conditions comprisehybridization in 5×SSPE, 1% SDS, 1×Denhardts solution at 65° C. andwashing in 2×SSC, 1% SDS and subsequently with 0.2×SSC at 65° C.

Also contemplated is a population of nucleic acid molecules comprising afirst nucleic acid molecule and a second nucleic acid molecule, wherein:

the first nucleic acid molecule is a nucleic acid molecule comprising anucleotide sequence encoding a heavy chain variable region (HCVR) or atau-binding fragment thereof of an anti-tau antibody, wherein said HCVRor fragment thereof comprises: (i) a framework derived from humanimmunoglobulin M65092 (SEQ ID NO. 71), (ii) an HCVR CDR1 comprising theamino acid sequence of SEQ ID NO: 1, (iii) an HCVR CDR2 comprising theamino acid sequence of SEQ ID NO: 2, and (iv) an HCVR CDR3 comprisingthe amino acid sequence of SEQ ID NO: 3, and wherein said HCVR orfragment thereof comprises an amino acid sequence that is at least 98%identical to the amino acid sequence of anyone of SEQ ID NO: RHA throughSEQ ID NO. RHM (i.e., SEQ ID NO. 13-25);

and wherein the second nucleic acid molecule comprises a nucleotidesequence encoding an LCVR or a tau-binding fragment thereof of ananti-tau antibody, wherein said LCVR or fragment thereof comprises: (i)a framework derived from human immunoglobulin X72449 (SEQ ID NO. 65),(ii) an LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 4,(iii) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO; 5,and (iv) an LCVR CDR3 comprising the amino acid sequence of SEQ ID NO:6.

Also contemplated is a population of nucleic acid molecules comprising afirst nucleic acid molecule and a second nucleic acid molecule, wherein:

a nucleic acid molecule comprising a nucleotide sequence encoding alight chain variable region (LCVR) or a tau-binding fragment thereof ofan anti-tau antibody, wherein said LCVR or fragment thereof comprises:(i) a framework derived from human immunoglobulin X72449 (SEQ ID NO.65), (ii) an LCVR CDR1 comprising the amino acid sequence of SEQ ID NO:4, (iii) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO:5, and (iv) an LCVR CDR3 comprising the amino acid sequence of SEQ IDNO: 6, and wherein said LCVR or fragment thereof comprises an amino acidsequence that is at least 98% identical to the amino acid sequence ofSEQ ID NO: 26, RKA;

and wherein the second nucleic acid molecule comprises a nucleotidesequence encoding an HCVR or a tau-binding fragment thereof of ananti-tau antibody, wherein said HCVR or fragment thereof comprises: (i)a framework derived from human immunoglobulin M65092 (SEQ ID NO. 71),(ii) an HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 1,(iii) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 2,and (iv) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO:3.

Also contemplated is a population of nucleic acid molecules comprising afirst nucleic acid molecule and a second nucleic acid molecule, wherein:

a nucleic acid molecule comprising a nucleotide sequence encoding alight chain variable region (LCVR) or a tau-binding fragment thereof ofan anti-tau antibody, wherein said LCVR or fragment thereof comprises:(i) a framework derived from human immunoglobulin X72449 (SEQ ID NO.65), (ii) an LCVR CDR1 comprising the amino acid sequence of SEQ ID NO:4, (iii) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO:5, and (iv) an LCVR CDR3 comprising the amino acid sequence of SEQ IDNO: 6, and wherein said LCVR or fragment thereof comprises an amino acidsequence that is at least 98% identical to the amino acid sequence ofSEQ ID NO: 27, RKB:

and wherein the second nucleic acid molecule comprises a nucleotidesequence encoding an HCVR or a tau-binding fragment thereof of ananti-tau antibody, wherein said HCVR or fragment thereof comprises: (i)a framework derived from human immunoglobulin M65092 (SEQ ID NO. 71),(ii) an HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 1,(iii) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 2,and (iv) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO:3.

Also contemplated are nucleic acid molecules encoding any of heavy chainvariable regions RHA-RHM (SEQ ID NOs 13-25), wherein those nucleic acidsequences are selected from any of SEQ ID NOs. 28-40, respectively, or43-55, respectively. Any of these nucleic acid molecules can be combinedwith the nucleic acid molecules of the following paragraph.

Also contemplated are nucleic acid molecules encoding any of lightchains RKA or RKB, wherein the nucleic acid molecules is selected fromSEQ ID NOs. 57 and 58, respectively.

All possible combinations of the nucleic acids disclosed above, thatencode an amino acid or tau-binding fragment thereof, a light chainvariable, a complete light chain, a heavy chain variable, a completeheavy chain, as disclosed herein, are contemplated.

In some examples of such nucleic acid populations, they comprise a firstnucleic acid molecule and a second nucleic acid molecule, wherein thefirst nucleic acid molecule comprises a nucleotide sequence encoding anHCVR or a tau-binding fragment thereof of an anti-tau antibody of SEQ IDNO. 14, RHB, and the second nucleic acid molecule comprises a nucleotidesequence encoding a LCVR or a tau-binding fragment thereof of SEQ ID NO.26, RKA.

In some examples of such populations, they comprise a first nucleic acidmolecule and a second nucleic acid molecule, wherein the first nucleicacid molecule comprises a nucleotide sequence encoding an HCVR or atau-binding fragment thereof of an anti-tau antibody of SEQ ID NO. 16,RHD, and the second nucleic acid molecule comprises a nucleotidesequence encoding a LCVR or a tau-binding fragment thereof of SEQ ID NO.26, RKA.

In some examples of such populations, they comprise a first nucleic acidmolecule and a second nucleic acid molecule, wherein the first nucleicacid molecule comprises a nucleotide sequence encoding an HCVR or atau-binding fragment thereof of an anti-tau antibody of SEQ ID NO. 17,RHE, and the second nucleic acid molecule comprises a nucleotidesequence encoding a LCVR or a tau-binding fragment thereof of SEQ ID NO.26, RKA.

In some examples of such populations, they comprise a first nucleic acidmolecule and a second nucleic acid molecule, wherein the first nucleicacid molecule comprises a nucleotide sequence encoding an HCVR or atau-binding fragment thereof of an anti-tau antibody of SEQ ID NO. 25,RHM, and the second nucleic acid molecule comprises a nucleotidesequence encoding a LCVR or a tau-binding fragment thereof of SEQ ID NO.26, RKA.

In some examples of such populations, they comprise a first nucleic acidmolecule and a second nucleic acid molecule, wherein the first nucleicacid molecule comprises a nucleotide sequence encoding an HCVR or atau-binding fragment thereof of an anti-tau antibody of SEQ ID NO. 16,RHD, and the second nucleic acid molecule comprises a nucleotidesequence encoding a LCVR or a tau-binding fragment thereof of SEQ ID NO.27, RKB.

In some examples of such populations, they comprise a first nucleic acidmolecule and a second nucleic acid molecule, wherein the first nucleicacid molecule comprises a nucleotide sequence encoding an HCVR or atau-binding fragment thereof of an anti-tau antibody of SEQ ID NO. 17,RHE, and the second nucleic acid molecule comprises a nucleotidesequence encoding a LCVR or a tau-binding fragment thereof of SEQ ID NO.27, RKB.

In some examples of such populations, they comprise a first nucleic acidmolecule and a second nucleic acid molecule, wherein the first nucleicacid molecule comprises a nucleotide sequence encoding an HCVR or atau-binding fragment thereof of an anti-tau antibody of SEQ ID NO. 14,RKB, and the second nucleic acid molecule comprises a nucleotidesequence encoding a LCVR or a tau-binding fragment thereof of SEQ ID NO.27, RKB.

Also contemplated is a set of vectors each comprising a nucleic acidencoding the heavy chain of the antibody or binding fragment asdescribed in the previous paragraphs of this section. Another set ofvectors contemplated are those comprising a nucleic acid encoding thelight chain of the antibody or binding fragment as described in any ofthe previous paragraphs of this section. Another set of vectorscontemplated are those comprising a nucleic acid encoding the lightchain of the antibody or binding fragment as described in any of theprevious paragraphs of this section and heavy chain of the antibody orbinding fragment as described in the previous paragraphs of thissection.

Host cells comprising any of these vectors are also contemplated. Insome embodiments, the host cell is prokaryotic. In other embodiments,the host cell is eukaryotic. A host cell comprising any of thepopulation of nucleic acid molecules described above are alsocontemplated.

In some embodiments of these host cells, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 14, RHB and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these host cells, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these host cells, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHE and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these host cells, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 25, RHM and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these host cells, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these host cells, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHM and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these host cells, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 14, RHB and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these host cells, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHM and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

Any of the above contemplated nucleic acids, antibodies, vectors, andhost cells, and their respective compositions is contemplated for use asa drug for:

the prevention or treatment of AD or another tauopathy;

treating Alzheimer's Disease or another tauopathy in a subject having,suspected of having, or being prone to have Alzheimer's Disease oranother tauopathy;

slowing progression of AD or another tauopathy in a subject having,suspected of having, or being prone to have Alzheimer's Disease oranother tauopathy;

ameliorating the symptoms of AD or a related in a subject having,suspected of having, or being prone to have Alzheimer's Disease oranother tauopathy; and

reducing the risk or delaying the onset of AD or another tauopathy in asubject having, suspected of having, or being prone to have Alzheimer'sDisease or another tauopathy.

Another aspect of the present disclosure is a method of producing anantibody or tau-binding fragment thereof that binds to human taucomprising culturing any of the host cells just described so that thenucleic acid is expressed and the antibody or tau-binding fragmentthereof produced.

In some embodiments of this method, the antibody and binding fragmentare also such that the sequence of the heavy chain variable region isSEQ ID NO. 14, RHB and the sequence of the light chain variable regionis SEQ ID NO. 26, RKA. Optionally, the antibody and binding fragment areof the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHE and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 25, RHM and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHM and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 14, RHB and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

Another aspect contemplates a method of treating Alzheimer's Disease oranother tauopathy in a subject having, suspected of having, or beingprone to have Alzheimer's Disease or another tauopathy, comprisingadministering to the subject a therapeutically effective amount of acomposition comprising any of the antibodies or binding fragmentsdescribed in this section.

In some embodiments of this method, the antibody and binding fragmentare also such that the sequence of the heavy chain variable region isSEQ ID NO. 14, RHB and the sequence of the light chain variable regionis SEQ ID NO. 26, RKA. Optionally, the antibody and binding fragment areof the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHE and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 25, RHM and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHM and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 14, RHB and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

Another aspect contemplates a method of promoting clearance of tauaggregates from the brain of a subject having, suspected of having, orbeing prone to have Alzheimer's Disease or another tauopathy, comprisingadministering to the subject a therapeutically effective amount of acomposition comprising any of the antibodies or binding fragmentsdescribed in this section.

In some embodiments of this method, the antibody and binding fragmentare also such that the sequence of the heavy chain variable region isSEQ ID NO. 14, RHB and the sequence of the light chain variable regionis SEQ ID NO. 26, RKA. Optionally, the antibody and binding fragment areof the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHE and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 25, RHM and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHM and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

Another aspect contemplates a method of slowing progression of AD oranother tauopathy in a subject having, suspected of having, or beingprone to have Alzheimer's Disease or another tauopathy, comprisingadministering to the subject a therapeutically effective amount of acomposition comprising any of the antibodies or binding fragmentsdescribed in this section.

In some embodiments of this method, the antibody and binding fragmentare also such that the sequence of the heavy chain variable region isSEQ ID NO. 14, RHB and the sequence of the light chain variable regionis SEQ ID NO. 26, RKA. Optionally, the antibody and binding fragment areof the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHE and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 25, RHM and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHM and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 14, RHB and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

Another aspect contemplates a method of ameliorating the symptoms of ADor a related in a subject having, suspected of having, or being prone tohave Alzheimer's Disease or another tauopathy, comprising administeringto the subject a therapeutically effective amount of a compositioncomprising any of the antibodies or binding fragments described in thissection.

In some embodiments of this method, the antibody and binding fragmentare also such that the sequence of the heavy chain variable region isSEQ ID NO. 14, RHB and the sequence of the light chain variable regionis SEQ ID NO. 26, RKA. Optionally, the antibody and binding fragment areof the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHE and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 25, RHM and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHM and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 14, RHB and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

Another aspect contemplates a method of treating, preventing, orreversing cognitive in a subject having, suspected of having, or beingprone to have Alzheimer's Disease or another tauopathy, comprisingadministering to the subject a therapeutically effective amount of acomposition comprising any of the antibodies or binding fragmentsdescribed in this section.

In some embodiments of this method, the antibody and binding fragmentare also such that the sequence of the heavy chain variable region isSEQ ID NO. 14, RHB and the sequence of the light chain variable regionis SEQ ID NO. 26, RKA. Optionally, the antibody and binding fragment areof the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHE and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 25, RHM and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHM and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 14, RHB and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

Another aspect contemplates a method of reducing the risk or delayingthe onset of AD or another tauopathy in a subject having, suspected ofhaving, or being prone to have Alzheimer's Disease or another tauopathy,comprising administering to the subject a therapeutically effectiveamount of a composition comprising any of the antibodies or bindingfragments described in this section.

In some embodiments of this method, the antibody and binding fragmentare also such that the sequence of the heavy chain variable region isSEQ ID NO. 14, RHB and the sequence of the light chain variable regionis SEQ ID NO. 26, RKA. Optionally, the antibody and binding fragment areof the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHE and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 25, RHM and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHM and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 14, RHB and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some embodiments, any of these compositions, antibodies, and/orbinding fragments is administered via injection. In other embodiments,any of these compositions, antibodies, and/or binding fragments isadministered via intravenous infusion.

Also contemplated are embodiments wherein any of these compositions,antibodies, and/or binding fragments is administered to said subject ata dose of antibody or binding fragment of 0.1 mg/kg of body weight to 20mg/kg of body weight and a frequency of between weekly and monthly,thereby treating the subject. In a preferred embodiment, the antibody orbinding fragment is administered at a dose of 0.1 mg/kg of body weightto 10 mg/kg of body weight. In some preferred embodiments, the antibodyor binding fragment is administered over a period of at least threemonths, preferably at least six months, likely at least twelve months,possibly for twenty four months, at any of these previous doses. Inanother embodiment, any of these compositions, antibodies, and/orbinding fragments is administered to said subject at a dose of antibodyor binding fragment of 0.1 mg/kg of body weight to 10 mg/kg of bodyweight and a frequency of between weekly and monthly, preferably everytwo weeks, thereby treating the subject. In another embodiment, any ofthese compositions, antibodies, and/or binding fragments is administeredto said subject at a dose of antibody or binding fragment of 0.01 mg/kgof body weight to 100 mg/kg of body weight and a frequency of betweenweekly and monthly, thereby treating the subject

In some embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 14, RHB, and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD, and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHE, and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 25, RHM, and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD, and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHM, and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In another aspect, any of the therapeutic methods just described in thissection further comprise monitoring the subject for treatmentprogression by at least one type of assessment selected from the groupconsisting of Mini-Mental State Exam (MMSE), Alzheimer's DiseaseAssessment Scale-cognitive (ADAS-COG), Clinician Interview-BasedImpression (CIBI), Neurological Test Battery (NTB), DisabilityAssessment for Dementia (DAD), Clinical Dementia Rating-sum of boxes(CDR-SOB), Neuropsychiatric Inventory (NPI), Positron EmissionTomography (PET Imaging) scan, and Magnetic Resonance Imaging (MRI)scan. In one embodiment, the type of assessment is a Neurological TestBattery (NTB). In another embodiment, the type of assessment is aMini-Mental State Exam (MMSE).

In some embodiments of this method, the antibody and binding fragmentare also such that the sequence of the heavy chain variable region isSEQ ID NO. 14, RHB and the sequence of the light chain variable regionis SEQ ID NO. 26, RKA. Optionally, the antibody and binding fragment areof the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHE and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 25, RHM and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHM and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some embodiments, the practice of any of these methods furthercomprises administering to said subject, concurrently or sequentially,an effective amount of at least one additional therapeutic agent.

In some embodiments of this method, the antibody and binding fragmentare also such that the sequence of the heavy chain variable region isSEQ ID NO. 14, RHB and the sequence of the light chain variable regionis SEQ ID NO. 26, RKA. Optionally, the antibody and binding fragment areof the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHE and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 25, RHM and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHM and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

For example, in some of these embodiments, the additional therapeuticagent is selected from the group consisting of an anti-apoptoticcompound, a metal chelator, an inhibitor of DNA repair,3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate (1,3PDS),a secretase activator, a beta-secretase inhibitor, a gamma-secretaseinhibitor, a beta-amyloid peptide, an anti-beta-amyloid antibody, aneurotransmitter, a beta-sheet breaker, an anti-inflammatory molecule,and a cholinesterase inhibitor; and any pharmaceutically acceptablesalts thereof.

In some other of these embodiments, the cholinesterase inhibitor istacrine, rivastigmine, donepezil, galantamine, or a nutritivesupplement; and any pharmaceutically acceptable salts thereof.

In some embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 14, RHB and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHE and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 25, RHM and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHM and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In yet some other of these embodiments, the additional therapeutic agentis selected from beta-amyloid peptides (e.g., N-terminal amyloid betapeptides), which might or might not be conjugated to other compounds,such as mutated diphtheria toxin; antibodies against beta-amyloid, suchas bapineuzumab, solaneuzumab, gantenerumab, crenezumab, ponezumab, andIVIG immunoglobulin, other immunization therapies targeting Abetaoligomers, compounds preventing the hyperphosphorylation of tau, acompound preventing tau oligomerization and aggregation or promotingdepolymerization of tau oligomers (e.g. methylthioninium, rember orLMTX) and other active and passive immunization therapies targetingpathological forms of tau (e.g. aggregates); and any pharmaceuticallyacceptable salts thereof.

In some of these other embodiments, the additional therapeutic agent isselected from amyloid-beta aggregation inhibitors (e.g., Tramiprosate),gamma-secretase inhibitors (e.g., semagacestat), and gamma-secretasemodulators (tarenflurbil); and any pharmaceutically acceptable saltsthereof.

In some embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 14, RHB and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHE and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 25, RHM and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHM and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some of these embodiments, the additional therapeutic agent isselected from acetylcholinesterase inhibitors (e.g., donepezil,rivastigmine, galantamine, tacrine, nutritive supplements),N-Methyl-D-aspartate (NMDA) receptor antagonists (e.g., memantine),inhibitors of DNA repair (e.g., pirenzepine or a metabolite thereof),transition metal chelators, growth factors, hormones, non-steroidalanti-inflammatory drugs (NSAID), antioxidants, lipid lowering agents,selective phosphodiesterase inhibitors, inhibitors of tau aggregation,inhibitors of protein kinases, inhibitors of anti-mitochondrialdysfunction drugs, neurotrophins, inhibitors of heat shock proteins,inhibitors of Lipoprotein-associated phospholipase A₂, memantine, ananti-apoptotic compound, a metal chelator, an inhibitor of DNA repair,3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate (1,3PDS),a secretase activator, a beta-secretase inhibitor, a gamma-secretaseinhibitor, a beta-amyloid peptide, a beta-amyloid antibody, aneurotransmitter, a beta-sheet breaker, an anti-inflammatory molecule;and any pharmaceutically acceptable salts thereof.

In some embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 14, RHB and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHE and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 25, RHM and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHM and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some of these embodiments, the additional therapeutic agent isselected from BACE inhibitors; muscarinic antagonists; cholinesteraseinhibitors; gamma secretase inhibitors; gamma secretase modulators;HMG-CoA reductase inhibitors; non-steroidal anti-inflammatory agents;N-methyl-D-aspartate receptor antagonists; anti-amyloid antibodies;vitamin E; nicotinic acetylcholine receptor agonists; CB1 receptorinverse agonists or CB1 receptor antagonists; an antibiotic; growthhormone secretagogues; histamine H3 antagonists; AMPA agonists; PDE4inhibitors; GABA_(A) inverse agonists; inhibitors of amyloidaggregation; glycogen synthase kinase beta inhibitors; promoters ofalpha secretase activity: PDE-10 inhibitors, cholesterol absorptioninhibitors, and any pharmaceutically acceptable salts thereof.

In some of these embodiments, the additional therapeutic agent is asecond antibody. In yet other embodiments, the second antibody isselected from bapineuzumab, solaneuzumab, gantenerumab, crenezumab,ponezumab, and IVIG immunoglobulin.

In some embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 14, RHB and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHE and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 25, RHM and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of these two methods, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHM and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

Also contemplated is a method of evaluating a subject having, suspectedof having, or being prone to have Alzheimer's Disease or anothertauopathy, the method comprising the step of detecting binding of any ofthe antibodies or tau-binding fragment described in this section to acomponent of a biological sample from the subject, wherein the detectionof binding to the biological sample is indicative of Alzheimer's Diseaseor another tauopathy in the subject. In some of these embodiments, thebiological sample is a biopsy, a CSF, blood, serum, or plasma sample.

In some embodiments of this method, the antibody and binding fragmentare also such that the sequence of the heavy chain variable region isSEQ ID NO. 14, RHB and the sequence of the light chain variable regionis SEQ ID NO. 26, RKA. Optionally, the antibody and binding fragment areof the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHE and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 25, RHM and the sequence of the light chainvariable region is SEQ ID NO. 26, RKA. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 16, RHD and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

In some other embodiments of this method, the antibody and bindingfragment are also such that the sequence of the heavy chain variableregion is SEQ ID NO. 17, RHM and the sequence of the light chainvariable region is SEQ ID NO. 27, RKB. Optionally, the antibody andbinding fragment are of the IgG1 or IgG4 isotype.

It is also contemplated that any of the therapeutic methods described inthe previous paragraphs can be practiced with an antibody or tau-bindingfragment, wherein the antibody and binding fragment are also such thatthe sequence of the heavy chain variable region is SEQ ID NO. 14, RHBand the sequence of the light chain variable region is SEQ ID NO. 26,RKA. Optionally, the antibody and binding fragment are of the IgG1 orIgG4 isotype.

It is also contemplated that any of the therapeutic methods described inthe previous paragraphs can be practiced with an antibody or tau-bindingfragment, wherein the antibody and binding fragment are also such thatthe sequence of the heavy chain variable region is SEQ ID NO. 16, RHDand the sequence of the light chain variable region is SEQ ID NO. 26,RKA. Optionally, the antibody and binding fragment are of the IgG1 orIgG4 isotype.

It is also contemplated that any of the therapeutic methods described inthe previous paragraphs can be practiced with an antibody or tau-bindingfragment, wherein the antibody and binding fragment are also such thatthe sequence of the heavy chain variable region is SEQ ID NO. 17, RHEand the sequence of the light chain variable region is SEQ ID NO. 26,RKA. Optionally, the antibody and binding fragment are of the IgG1 orIgG4 isotype.

It is also contemplated that any of the therapeutic methods described inthe previous paragraphs can be practiced with an antibody or tau-bindingfragment, wherein the antibody and binding fragment are also such thatthe sequence of the heavy chain variable region is SEQ ID NO. 25, RHMand the sequence of the light chain variable region is SEQ ID NO. 26,RKA. Optionally, the antibody and binding fragment are of the IgG1 orIgG4 isotype.

It is also contemplated that any of the therapeutic methods described inthe previous paragraphs can be practiced with an antibody or tau-bindingfragment, wherein the antibody and binding fragment are also such thatthe sequence of the heavy chain variable region is SEQ ID NO. 16, RHDand the sequence of the light chain variable region is SEQ ID NO. 27,RKB. Optionally, the antibody and binding fragment are of the IgG1 orIgG4 isotype.

It is also contemplated that any of the therapeutic methods described inthe previous paragraphs can be practiced with an antibody or tau-bindingfragment, wherein the antibody and binding fragment are also such thatthe sequence of the heavy chain variable region is SEQ ID NO. 17, RHMand the sequence of the light chain variable region is SEQ ID NO. 27,RKB. Optionally, the antibody and binding fragment are of the IgG1 orIgG4 isotype.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic of six isoforms of human tau

FIG. 2: Schematic functional map of human tau (2N4R); discloses “VQIINK”and “VQIVYK” as SEQ ID NOS 147 and 146, respectively.

FIG. 3: Protein (SEQ ID NO: 8) and DNA sequence (SEQ ID NO: 91) of DC8E8Kappa Light Chain Variable Region

FIG. 4: Protein (SEQ ID NO: 7) and DNA Sequence (SEQ ID NO: 90) of DC8E8Heavy Chain Variable Region

FIG. 5: DC8E8 Kappa Light Chain Germ Line Analysis (SEQ ID NOS 167 and168, respectively, in order of appearance)

FIG. 6: DC8E8 Heavy Chain Germ Line Analysis (SEQ ID NOS 66 and 169,respectively, in order of appearance)

FIG. 7: Binding of chimeric and murine DC8E8 to Tau 151-391/4R

FIG. 8: DC8E8 Heavy Chain Humanization Strategy. Structurally importantresidues (Proline, Cysteine and Asparagine) are highlighted by boldtypeface and italicised. Bold underlined residues indicateback-translations to the Mouse Residue. CDR regions are in lowercaseletters and indicated by dots in the table header. Residues within 4 Åof a CDR are indicated by stars in the table header. FIG. 8 disclosesSEQ ID NOS 7, 71 and 13-25, respectively, in order of appearance. Notethat SEQ ID NO. 71 (accession number M65092 in the IMGT database) isshown without the leader peptide MDWTWRFLFVVAAVTGVQS (SEQ ID NO. 174).

FIG. 9: DC8E8 Kappa Light Chain Humanization Strategy. Structurallyimportant residues (Proline, Cysteine and Asparagine) are highlighted bybold typeface and italicised. Bold underlined residues indicateback-translations to the Mouse Residue. CDR regions are in lowercaseletters and indicated by dots in the table header. Residues within 4 Åof a CDR are indicated by stars in the table header. FIG. 9 disclosesSEQ ID NOs. 8, 65 and 26-27, respectively, in order of appearance. Notethat SEQ ID NO. 65 (accession number X72449 in the IMGT database) isshown without the leader peptide QLLGLLMLWVSGSSG (SEQ ID NO. 175).

FIG. 10: Binding of humanized and chimeric DC8E8 to Tau 151-391/4R.Comparison of antibody binding by antibodies encoded by combinations ofDC8E8 RHA or DC8E8 RHB co-expressed with DC8E8 RKA or DC8E8 RKB with thefully chimeric antibody.

FIG. 11: Binding of humanized and chimeric DC8E8 to Tau 151-391/4R: RKAVersions.

FIG. 12: Binding of humanized and chimeric DC8E8 to Tau 151-391/4R: RKBVersions.

FIG. 13: Binding of humanized and chimeric DC8E8 to Tau 151-391/4R: VkVersions (with chimeric light chain).

FIG. 14: Binding of humanized and chimeric DC8E8 to Tau 151-391/4R: RHMVersions.

FIG. 15: Thermal stability of candidate humanized DC8E8 antibodies:Purified antibodies were heated for 10 min at the indicated temperature,then cooled to 4° C. before performing the Tau Binding ELISA.

FIG. 16: Thermal shift analysis of the purified humanized candidateDC8E8 antibodies: Determination of the purified candidate antibodies Tmand comparison to chimeric and murine DC8E8.

FIG. 17: Binding kinetics of DC8E8 antibodies analysed by surfaceplasmon resonance: KD determination: Murine DC8E8.

FIG. 18: Binding kinetics of DC8E8 antibodies analysed by surfaceplasmon resonance: K_(D) determination: Chimeric DC8E8

FIG. 19: Binding kinetics of DC8E8 antibodies analysed by surfaceplasmon resonance: K_(D) determination: Humanized DC8E8 RHD/RKA (AX004)

FIG. 20: Binding kinetics of DC8E8 antibodies analysed by surfaceplasmon resonance: K_(D) determination: Humanized DC8E8 RHE/RKA (AX005)

FIG. 21: Binding kinetics of DC8E8 antibodies analysed by surfaceplasmon resonance: K_(D) determination: Humanized DC8E8 RHD/RKB (AX016)

FIG. 22: Binding kinetics of DC8E8 antibodies analysed by surfaceplasmon resonance: K_(D) determination: Humanized DC8E8 RHE/RKB (AX017);

FIG. 23: Biacore Results Summary

FIG. 24: Fully humanized antibody candidates aggregation analysis: A.AX004 (DA); B. AX005 (EA); C. AX016 (DB); D. AX017 (EB).

FIG. 25: Purified humanized antibody candidates of DC8E8 assessed forsolubility: Solubility Profile during concentration.

FIG. 26: Purified humanized antibody candidates of DC8E8 assessed forsolubility: Solubility Profile during concentration: Binding activityafter concentration.

FIG. 27: Freeze/thaw stress analysis of humanized candidate antibodies.

FIG. 28: Heat induced stress analysis of fully humanized antibodycandidates: A. AX004 (DA); B. AX005 (EA); C. AX0016 (DB); D. AX017 (EB).

FIG. 29: Binding of (A) chimeric DC8E8 and (B) mouse DC8E8 tomisdisordered truncated tau 151-391/4R and physiological tau 2N4R asdetermined by ELISA. (C) EC50 values of chimeric and mouse antibody foranalyzed tau proteins.

FIG. 30: Mouse and chimeric DC8E8 equilibrium association bindingconstants to misdisordered truncated tau 151-391/4R and full lengthphysiological tau 2N4R were determined by surface plasmon resonance.

FIG. 31: Binding of (A) chimeric DC8E8 and (B) mouse DC8E8 to taupeptides derived from repeat domains of protein tau, determined byELISA. (C) EC50 values of chimeric and mouse antibody for analyzed taupeptides.

FIG. 32: Chimeric DC8E8 inhibits pathological tau-tau interaction in invitro tau fibrillization assay. Misdisordered truncated tau 151-391/4Rwas induced by heparin to undergo a conformational change and fibrilize,extent of which was measured by Thioflavin T fluorescence; chimericDC8E8 was tested for its ability to prevent the pathologicalconformational change and tau-tau interaction.

FIG. 33: Binding of humanized leads of DC8E8 (IgG4 isotype) and chimericDC8E8 to misdisordered tau 151-391/4R and full length physiological tau2N4R, as determined by ELISA. (A) Binding of humanized antibody AX004;(B) Binding of humanized antibody AX005; (C) Binding of humanizedantibody AX016; (D) Binding of humanized antibody AX017. (E) Binding ofchimeric DC8E8. (F) EC50 values of chimeric DC8E8 and humanized leadsAX004, AX005, AX016, AX017 to the analyzed tau proteins.

FIG. 34: Binding of humanized leads of DC8E8 (IgG1 isotype) tomisdisordered tau 151-391/4R and full length physiological tau 2N4R, asdetermined by ELISA (A) Binding of humanized antibody AX004; (B) Bindingof humanized antibody AX005; (C) Binding of humanized antibody AX016;(D) Binding of humanized antibody AX017. (E) EC50 values of humanizedleads AX004, AX005, AX016, AX017 to the analyzed tau proteins.

FIG. 35: Surface plasmon resonance (SPR) to characterize humanized DC8E8leads AX004, AX005, AX016, AX017, binding to mis-disorderedtau151-391/4R and full length 2N4R. (A) IgG4 versions, (B) IgG1versions.

FIG. 36: Binding of humanized antibodies (of IgG4 isotype) to taupeptides derived from the microtubule-binding repeat region of proteintau, as determined by ELISA. (A) Binding of humanized antibody AX004;(B) Binding of humanized antibody AX005; (C) Binding of humanizedantibody AX016; (D) Binding of humanized antibody AX017. (E) Binding ofchimeric DC8E8. (F) EC50 values of chimeric DC8E8 and humanized versionAX004, AX005, AX016, AX017 for analyzed tau proteins.

FIG. 37: Binding of humanized antibodies (of IgG1 isotype) to taupeptides derived from the microtubule-binding repeat region of proteintau, as determined by ELISA. (A) Binding of humanized antibody AX004;(B) Binding of humanized antibody AX005; (C) Binding of humanizedantibody AX016; (D) Binding of humanized antibody AX017. (E) EC50 valuesof chimeric DC8E8 and humanized version AX004, AX005, AX016, AX017 foranalyzed tau proteins.

FIG. 38: Humanized DC8E8 leads inhibit pathological tau-tau interactionin fluorescence-based tau fibrillization assay. Mis-disordered tau151-391/4R was induced to undergo a conformational change and fibrilizeas measured by Thioflavin T fluorescence; humanized antibodies wereadded to the fibrillization reaction and tested for their ability toprevent the pathological conformational change. All tested humanizedantibodies are capable of inhibiting pathological tau-tau aggregation.(A) Inhibition of pathological aggregation induced by humanized DC8E8leads of IgG4 isotype and (B) inhibition of pathological aggregationinduced by humanized antibodies of IgG1 isotype.

FIG. 39: Immunohistochemical staining of Alzheimer's disease brain usinghumanized DC8E8 antibodies (IgG1) Both DC8E8 (A) and chimeric DC8E8 (B)recognized high load of neurofibrillary pathology in the human ADhippocampus (CA1). Humanized antibodies AX004 (C) and AX016 (E)displayed the same staining pattern as DC8E8. In general humanizedantibodies AX005 (D) and AX017 (F) recognized less pathologicalstructures in AD brain. Tool bar: 100 μm

FIG. 40: Immunohistochemical staining of Alzheimer's disease brain usinghumanized DC8E8 antibodies (IgG4) Both DC8E8 (A) and chimeric DC8E8 (B)recognized high load of neurofibrillary pathology in the human ADhippocampus (CA1). Humanized antibodies AX004 (C) and AX016 (E)displayed the same staining pattern as DC8E8. In general humanizedantibodies AX005 (D) and AX017 (F) recognized less pathologicalstructures in AD brain. Tool bar: 100 μm

FIG. 41: Immunohistochemical staining of FTDP-17 brain (tau mutation atR406W) using humanized DC8E8 antibodies (IgG1) Both DC8E8 (A) andchimeric DC8E8 (B) recognized high load of neurofibrillary pathology inthe human entorhinal cortex. Humanized antibodies AX004 (C) and AX016(E) displayed the similar staining pattern as DC8E8. In generalhumanized antibodies AX005 (D) and AX017 (F) recognized lesspathological structures in FTDP-17 brain. Tool bar: 100 μm

FIG. 42: Immunohistochemical staining of FTDP-17 brain (tau mutation atR406W) using humanized DC8E8 antibodies (IgG4) Both DC8E8 (A) andchimeric DC8E8 (B) recognized high load of neurofibrillary pathology inthe human entorhinal cortex. Humanized antibodies AX004 (C) and AX016(E) displayed the same staining pattern as DC8E8. AX005 (D) and AX017(F) did not recognize any pathological structures in FTDP-17 brain. Toolbar: 100 μm

FIG. 43: Immunohistochemical staining of corticobasal degeneration usinghumanized DC8E8 antibodies (IgG1) Both DC8E8 (A) and chimeric DC8E8 (B)recognized high number of glial tau pathology in the human nucleuscaudatus. Humanized antibodies AX004 (C) and AX016 (E) displayed thesame staining pattern as DC8E8. In general humanized antibodies AX005(D) and AX017 (F) recognized less pathological structures in CBD brain,however the intensity of staining is comparable to DC8E8. Tool bar: 50μm

FIG. 44: Immunohistochemical staining of corticobasal degeneration usinghumanized DC8E8 antibodies (IgG4) Both DC8E8 (A) and chimeric DC8E8 (B)recognized high number of glial tau pathology in the human nucleuscaudatus. Humanized antibodies AX004 (C) and AX016 (E) displayed thesame staining pattern as DC8E8. In general humanized antibodies AX005(D) and AX017 (F) recognized less pathological structures in CBD brain,however the intensity of staining is comparable to DC8E8. Tool bar: 50μm

FIG. 45: Immunohistochemical staining of progressive supranuclear palsyusing humanized DC8E8 antibodies (IgG1) Both DC8E8 (A) and chimericDC8E8 (B) recognized high number of glial tau pathology in the humannucleus caudatus. Humanized antibodies AX004 (C) and AX016 (E) displayedthe same staining pattern as DC8E8. In general humanized antibodiesAX005 (D) and AX017 (F) recognized slightly less pathologicalstructures. The intensity of staining is comparable to DC8E8. Tool bar:50 μm

FIG. 46: Immunohistochemical staining of progressive supranuclear palsyusing humanized DC8E8 antibodies (IgG4) Both DC8E8 (A) and chimericDC8E8 (B) recognizes high number of glial tau pathology in the humannucleus caudatus. Humanized antibodies AX004 (C) and AX016 (E) displayedthe same staining pattern as DC8E8. In general humanized antibodiesAX005 (D) and AX017 (F) recognized slightly less pathologicalstructures. The intensity of staining is comparable to DC8E8. Tool bar:50 μm

The amino acid sequences corresponding to the human tau isoforms aregiven in SEQ ID NOs. 151-156, respectively, in order of appearance:

SEQ ID NO: 151 (2N4R): MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTDAGLKESPLQT PTEDGSEEPG SETSDAKSTP TAEDVTAPLVDEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAGHVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPPGQKGQANATR IPAKTPPAPK TPPSSGEPPK SGDRSGYSSPGSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAKSRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIINKKLDLSNVQSK CGSKDNIKHV PGGGSVQIVY KPVDLSKVTSKCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNITHVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPVVSGDTSPRHLSN VSSTGSIDMV DSPQLATLAD EVSASLAKQG L SEQ ID NO: 152 (1N4R):MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTDAGLKESPLQT PTEDGSEEPG SETSDAKSTP TAEAEEAGIGDTPSLEDEAA GHVTQARMVS KSKDGTGSDD KKAKGADGKTKIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPPKSGDRSGYSS PGSPGTPGSR SRTPSLPTPP TREPKKVAVVRTPPKSPSSA KSRLQTAPVP MPDLKNVKSK IGSTENLKHQPGGGKVQIIN KKLDLSNVQS KCGSKDNIKH VPGGGSVQIVYKPVDLSKVT SKCGSLGNIH HKPGGGQVEV KSEKLDFKDRVQSKIGSLDN ITHVPGGGNK KIETHKLTFR ENAKAKTDHGAEIVYKSPVV SGDTSPRHLS NVSSTGSIDM VDSPQLATLA DEVSASLAKQ GLSEQ ID NO: 153 (2N3R): MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTDAGLKESPLQT PTEDGSEEPG SETSDAKSTP TAEDVTAPLVDEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAGHVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPPGQKGQANATR IPAKTPPAPK TPPSSGEPPK SGDRSGYSSPGSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAKSRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIVYKPVDLSKVTSK CGSLGNIHHK PGGGQVEVKS EKLDFKDRVQSKIGSLDNIT HVPGGGNKKI ETHKLTFREN AKAKTDHGAEIVYKSPVVSG DTSPRHLSNV SSTGSIDMVD SPQLATLADE VSASLAKQGLSEQ ID NO: 154 (0N4R): MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTDAGLKAEEAGI GDTPSLEDEA AGHVTQARMV SKSKDGTGSDDKKAKGADGK TKIATPRGAA PPGQKGQANA TRIPAKTPPAPKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTPPTREPKKVAV VRTPPKSPSS AKSRLQTAPV PMPDLKNVKSKIGSTENLKH QPGGGKVQII NKKLDLSNVQ SKCGSKDNIKHVPGGGSVQI VYKPVDLSKV TSKCGSLGNI HHKPGGGQVEVKSEKLDFKD RVQSKIGSLD NITHVPGGGN KKIETHKLTFRENAKAKTDH GAEIVYKSPV VSGDTSPRHL SNVSSTGSID MVDSPQLATL ADEVSASLAK QGLSEQ ID NO: 155 (1N3R): MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTDAGLKESPLQT PTEDGSEEPG SETSDAKSTP TAEAEEAGIGDTPSLEDEAA GHVTQARMVS KSKDGTGSDD KKAKGADGKTKIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPPKSGDRSGYSS PGSPGTPGSR SRTPSLPTPP TREPKKVAVVRTPPKSPSSA KSRLQTAPVP MPDLKNVKSK IGSTENLKHQPGGGKVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVKSEKLDFKDRV QSKIGSLDNI THVPGGGNKK IETHKLTFRENAKAKTDHGA EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV DSPQLATLAD EVSASLAKQG LSEQ ID NO: 156 (0N3R): MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTDAGLKAEEAGI GDTPSLEDEA AGHVTQARMV SKSKDGTGSDDKKAKGADGK TKIATPRGAA PPGQKGQANA TRIPAKTPPAPKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTPPTREPKKVAV VRTPPKSPSS AKSRLQTAPV PMPDLKNVKSKIGSTENLKH QPGGGKVQIV YKPVDLSKVT SKCGSLGNIHHKPGGGQVEV KSEKLDFKDR VQSKIGSLDN ITHVPGGGNKKIETHKLTFR ENAKAKTDHG AEIVYKSPVV SGDTSPRHLSNVSSTGSIDM VDSPQLATLA DEVSASLAKQ GL

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “affinity” refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Theaffinity of a molecule X for its partner Y can generally be representedby the equilibrium dissociation constant (K_(D)) (or its inverseequilibrium association constant, K_(A)). Affinity can be measured bycommon methods known in the art, including those described herein. See,for example, Pope M E, Soste M V, Eyford B A, Anderson N L, Pearson T W.(2009) J Immunol Methods, 341(1-2):86-96 and methods described herein.Specific illustrative and exemplary embodiments for measuring bindingaffinity are described below.

The term “amino acid” refers to naturally occurring, modified, andsynthetic amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified,e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Aminoacid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., an alpha carbonthat is bound to a hydrogen, a carboxyl group, an amino group, and an Rgroup, e.g., homoserine, norleucine, methionine sulfoxide, methioninemethyl sulfonium. Such analogs have modified R groups (e.g., norleucine)or modified peptide backbones, but retain the same basic chemicalstructure as a naturally occurring amino acid. Amino acid mimeticsrefers to chemical compounds that have a structure that is differentfrom the general chemical structure of an amino acid, but that functionin a manner similar to a naturally occurring amino acid. Suitable aminoacids include, without limitation, both the D- and L-isomers of the 20common naturally occurring amino acids found in peptides as well as thenaturally occurring and unnaturally occurring amino acids prepared byorganic synthesis or other metabolic routes. Examples of suchunnaturally occurring amino acids include, but are not limited to,N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, andO-phosphotyrosine. Modified amino acids include, but are not limited to,hydroxyproline, pyroglutamate, gamma-carboxyglutamate, O-phosphoserine,azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid,beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyricacid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyricacid, 3-aminoisobutyric acid, 2-aminopimelic acid,tertiary-butylglycine, 2,4-diaminoisobutyric acid, desmosine,2,2′-diaminopimelic acid, 2,3-diaminoproprionic acid, N-ethylglycine,N-methylglycine, N-ethylasparagine, homoproline, hydroxylysine,allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine,allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine,N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline,norleucine, ornithine, pentylglycine, pipecolic acid and thioproline.The term amino acid also includes naturally occurring amino acids thatare metabolites in certain organisms but are not encoded by the geneticcode for incorporation into proteins. Such amino acids include, but arenot limited to, ornithine, D-ornithine, and D-arginine.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express Fc.gamma.RIII only, whereas monocytes expressFc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR expression onhematopoietic cells in summarized is Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of amolecule of interest, an in vitro ADCC assay, such as that described inU.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337 may be performed.Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in a animal model such as that disclosed inClynes et al. PNAS (USA) 95:652-656 (1998).

A “back mutation” is a mutation introduced in a nucleotide sequencewhich encodes a humanized antibody, the mutation results in an aminoacid corresponding to an amino acid in the parent antibody (e.g., donorantibody, for example, a murine antibody). Certain framework residuesfrom the parent antibody may be retained during the humanization of theantibodies of the disclosure in order to substantially retain thebinding properties of the parent antibody, while at the same timeminimizing the potential immunogenicity of the resultant antibody. Inone embodiment disclosed herein, the parent antibody is of mouse origin.For example, the back mutation changes a human framework residue to aparent murine residue. Examples of framework residues that may be backmutated include, but are not limited to, canonical residues, interfacepacking residues, unusual parent residues which are close to the bindingsite, residues in the “Vernier Zone” (which forms a platform on whichthe CDRs rest) (Foote & Winter, 1992, J. Mol. Biol. 224, 487-499), andthose close to CDR H3.

The term “chimeric” antibodies refers to antibodies in which a portionof the heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass (e.g., chimerichumanized, class-switched antibodies), while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).In one embodiment, the term “chimeric antibody” refers to a monoclonalantibody comprising a variable region, i.e., binding region, from onesource or species and at least a portion of a constant region derivedfrom a different source or species, usually prepared by recombinant DNAtechniques. Chimeric antibodies comprising a murine variable region anda human constant region are sometimes preferred. Such murine/humanchimeric antibodies are the product of expressed immunoglobulin genescomprising DNA segments encoding murine immunoglobulin variable regionsand DNA segments encoding human immunoglobulin constant regions. Otherforms of “chimeric antibodies” encompassed by the present invention arethose in which the class or subclass has been modified or changed fromthat of the original antibody. Such “chimeric” antibodies are alsoreferred to as “class-switched antibodies.” Methods for producingchimeric antibodies involve conventional recombinant DNA and genetransfection techniques now known in the art. See, e.g., Morrison, S.L., et al., Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; U.S. Pat.Nos. 5,202,238 and 5,204,244.

“Competitive binding” is determined in an assay in which theimmunoglobulin/antibody/binding fragment under testing inhibits specificbinding of a reference antibody to a common antigen, such as tau (e.g.,tau 151-391/4R). Numerous types of competitive binding assays are known,for example: solid phase direct or indirect radioimmunoassay (RIA),solid phase direct or indirect enzyme immunoassay (EIA), sandwichcompetition assay (see Stahli et al., Methods in Enzymology 9:242(1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J.Immunol. 137:3614 (1986)); solid phase direct labeled assay, solid phasedirect labeled sandwich assay (see Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Press (1988)); solid phase directlabel RIA using I¹²⁵ label (see Morel et al., Mol. Immunol. 25(1):7(1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology176:546 (1990)); and direct labeled RIA. (Moldenhauer et al., Scand. J.Immunol. 32:77 (1990)). Typically, such an assay involves the use ofpurified antigen bound to a solid surface or cells bearing either ofthese, an unlabeled test immunoglobulin and a labeled referenceimmunoglobulin. Competitive inhibition is measured by determining theamount of label bound to the solid surface or cells in the presence ofthe test immunoglobulin. Usually the test immunoglobulin is present inexcess. Usually, when a competing antibody is present in excess, it willinhibit specific binding of a reference antibody to a common antigen byat least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more.

“Complement dependent cytotoxicity” or “CDC” refers to the ability of amolecule to lyse a target in the presence of complement. The complementactivation pathway is initiated by the binding of the first component ofthe complement system (C1q) to a molecule (e.g. an antibody) complexedwith a cognate antigen. To assess complement activation, a CDC assay,e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163(1996), may be performed.

The term “conjugation,” as used herein, refers to a bond or chemicalmoiety formed from a chemical reaction between a functional group of afirst molecule (e.g., an antibody) with a functional group of a secondmolecule (e.g., a therapeutic agent or drug). Such bonds include, butare not limited to, covalent linkages and non-covalent bonds, while suchchemical moieties include, but are not limited to, esters, carbonates,imines phosphate esters, hydrazones, acetals, orthoesters, peptidelinkages, and oligonucleotide linkages. “Hydrolytically stable linkages”means that the linkages are substantially stable in water and do notreact with water at useful pH values, including but not limited to,under physiological conditions for an extended period of time.“Hydrolytically unstable or degradable linkages” means that the linkagesare degradable in water or in aqueous solutions, including for example,blood. “Enzymatically unstable or degradable linkages” means that thelinkages are degraded by one or more enzymes. By way of example only,certain PEG and related polymers include degradable linkages in thepolymer backbone or in a linker group between the PEG polymer backboneand one or more of the terminal functional groups of protein,polypeptide or peptide provided herein. Such degradable linkagesinclude, but are not limited to, ester linkages formed by the reactionof PEG carboxylic acids or activated PEG carboxylic acids with alcoholgroups on a biologically active agent, wherein such ester groupsgenerally hydrolyze under physiological conditions to release thebiologically active agent. Other hydrolytically degradable linkagesinclude but are not limited to carbonate linkages; imine linkagesresulted from reaction of an amine and an aldehyde; phosphate esterlinkages formed by reacting an alcohol with a phosphate group; hydrazonelinkages which are reaction product of a hydrazide and an aldehyde;acetal linkages that are the reaction product of an aldehyde and analcohol; orthoester linkages that are the reaction product of a formateand an alcohol; peptide linkages formed by an amine group, including butnot limited to, at an end of a polymer such as PEG, and a carboxyl groupof a peptide; and oligonucleotide linkages formed by a phosphoramiditegroup, including but not limited to, at the end of a polymer, and a 5′hydroxyl group of an oligonucleotide

The term “DC8E8” refers to an antibody described in WO2013/041962 andproduced by the hybridoma deposited under American Type CultureCollection Patent Deposit no. PTA-11994. Disclosed in WO2013/041962 isthe discovery that antibodies (e.g., DC8E8) that specifically bind toone or more of four previously unidentified functional regions of tauselected from 268-HQPGGG-273 (SEQ ID NO: 148) (within 1^(st) repeatdomain of tau protein), 299-HVPGGG-304 (SEQ ID NO: 149) (within 2^(nd)repeat domain of tau protein), 330-HKPGGG-335 (SEQ ID NO: 150) (within3^(rd) repeat domain of tau protein), and 362-HVPGGG-367 (SEQ ID NO:149) (within 4^(th) repeat domain of tau protein) are capable ofinhibiting formation of pathological tau aggregates, and of detectingvarious pathological forms of tau, some of which are the earliest formedin the disease (e.g., pathological monomers). Hybridomas producedagainst human misdisordered tau II (Tau 151-391/4R), which is alsoreferred in this application as tauΔ(1-150; 392-441)/4R, were screenedfor the production of monoclonal antibodies specific to human pairedhelical filaments (PHFs) both by immunohistochemistry (IHC) andEnzyme-linked Immuno Assays (ELISAs). The resulting set included mousemonoclonal antibody (mAb) DC8E8, which is of the IgG1 subclass. Epitopemapping of DC8E8 revealed it to bind four previously unidentifiedepitopes on human tau. Moreover, further functional analysis of DC8E8revealed that each epitope represents a distinct functional regionwithin tau. These regions, which were described as novel targets for ADdiagnosis and therapy, are comprised within functional regions of tauselected from 268-HQPGGG-273 (SEQ ID NO: 148) (within 1^(st) repeatdomain of tau protein), 299-HVPGGG-304 (SEQ ID NO: 149) (within 2^(nd)repeat domain of tau protein), 330-HKPGGG-335 (SEQ ID NO: 150) (within3^(rd) repeat domain of tau protein), and 362-HVPGGG-367 (SEQ ID NO:149) (within 4^(th) repeat domain of tau protein).

The terms “diagnosing” and “diagnosis” as used herein refer to methodsby which the skilled artisan can estimate and even determine whether ornot a subject is suffering from a given disease or condition, in thiscase Alzheimer's disease and related tauopathies. The skilled artisanoften makes a diagnosis on the basis of one or more diagnosticindicators, such as for example a biomarker, the amount (includingpresence or absence) of which is indicative of the presence, severity,or absence of the condition.

Along with diagnosis, clinical disease monitoring and prognosis is alsoan area of great concern and interest. It is important to know the stageand rapidity of advancement of the AD in order to plan the mosteffective therapy. If a more accurate prognosis can be made, appropriatetherapy, and in some instances less severe therapy for the patient canbe chosen. Measurement of pathological tau levels as disclosed hereincan be useful in order to categorize subjects according to advancementof AD who will benefit from particular therapies and differentiate fromother subjects where alternative or additional therapies can be moreappropriate.

For example, DC8E8 is able to discriminate between preclinical AD,clinically incipient AD and fully developed final stage AD (seeWO20131041962). DC8E8 displayed staining of early stages (tau monomers,dimers) of pathological tau in human preclinical AD—Braak's Stage I. Theantibody recognized the stage of pathological tau oligomers and thestage of pathological tau polymers (tangles). In fully developedAlzheimer's disease (final stage—Braak's Stage VI), DC8E8 recognizedmainly pathological tau polymers in forms of the neurofibrillarytangles, neuritic plaques and neuritic threads. DC8E8 recognized alldevelopmental stages of tangle formation in Alzheimer's disease. DC8E8recognized early developmental stages of tangle formation—monomeric,dimeric and early oligomeric stage, and late oligomeric, pre-tanglestage, as well as late developmental stages of pathological taupolymers—intracellular and extracellular neurofibrillary tangles.

Accordingly, “diagnosing” or “making a diagnosis,” as used herein, isfurther inclusive of making a prognosis, which can provide forpredicting a clinical outcome (with or without medical treatment),selecting an appropriate treatment (or whether treatment would beeffective), or monitoring a current treatment and potentially changingthe treatment, based on the measure of pathological tau levels.

Antibody “effector functions” refer to those biological activitiesattributable to an Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; and down regulation of cell surfacereceptors (e.g. B cell receptor; BCR).

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an immunoglobulin or antibody (or antigen bindingfragment thereof) specifically binds. Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids, often in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, G. E. Morris, Ed. (1996). A “conformationalepitope” is an epitope to which the antibody or tau-binding fragmentthereof binds in a conformational-specific manner. In the case ofprotein-based epitopes, the binding can depend on theepitope-carrying-protein's secondary, tertiary, or quaternary structure.In other words, the antibody binds in a structure-specific manner, atertiary-structure-specific manner, or a quaternary-structure-specificmanner. A conformational epitope is one that is present in pathologicaltau (e.g., present in Tau 151-391/4R).

The antibodies and tau-binding fragments described herein have as their“epitope” any one or more of the following four tau sites, some or allof which are conformational epitopes: 268-HQPGGG-273 (SEQ ID NO: 148)(within 1^(st) repeat domain of tau protein), 299-HVPGGG-304 (SEQ ID NO:149) (within 2^(nd) repeat domain of tau protein), 330-HKPGGG-335 (SEQID NO: 150) (within 3^(rd) repeat domain of tau protein), and362-HVPGGG-367 (SEQ ID NO: 149) (within 4^(th) repeat domain of tauprotein). These epitopes are also referred herein as QT1, QT2, QT3, andQT4, respectively. DC8E8 binds to all of QT1-QT4; in fact, the epitopeof DC8E8 is HXPGGG, wherein X is any amino acid (SEQ ID NO. 157).

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily (fragment crystalizable). Pepsin treatment yields anF(ab′)₂ fragment that has two antigen-binding sites and is still capableof cross-linking antigen. “Fv” is that portion of the heavy chain thatis included in a Fab fragment. Any of these fragments can also beproduced recombinantly. The Fc portion of an antibody is associated withthe antibody's effector functions, including antibody-dependent cellularcytotoxicity (ADCC) and complement-dependent cytotoxicity orphagocytosis. Alterations (e.g., mutations or glycosylation changes) inthe Fc region of an antibody can be used to modulate any of its effectorfunctions as well as increase its serum half-life and otherpharmacokinetic properties.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear at least one free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The term “Fc”, as used herein, includes the polypeptide comprising theconstant region of an antibody excluding the first constant regionimmunoglobulin domain. Thus Fc refers to the last two constant regionimmunoglobulin domains of IgA, IgD, and IgG, and the last three constantregion immunoglobulin domains of IgE and IgM, and the flexible hingeN-terminal to these domains. For IgA and IgM, Fc may include the Jchain. For IgG, Fc comprises immunoglobulin domains C gamma 2 and Cgamma 3 (Cgamma2 and Cgamma3) and the hinge between C gamma 1(Cgamma1)and C gamma 2 (Cgamma2). Although the boundaries of the Fc region mayvary, the human IgG heavy chain Fc region is usually defined to compriseresidues C226 or P230 to its carboxyl-terminus, wherein the numbering isaccording to the EU numbering system. For human IgG1 the Fc region isherein defined, in one embodiment, to comprise residue P232 to itscarboxyl-terminus, wherein the numbering is according to the EUnumbering system (Edelman G M et al., (1969) Proc Natl Acad Sci USA,63(1): 78-85). The C-terminal lysine (residue 447 according to the EUnumbering system) of the Fc region may be removed, for example, duringproduction or purification of the antibody, or by recombinantlyengineering the nucleic acid encoding a heavy chain of the antibody.Accordingly, a composition of intact antibodies may comprise antibodypopulations with all K447 residues removed, antibody populations with noK447 residues removed, and antibody populations having a mixture ofantibodies with and without the K447 residue. Fc may refer to thisregion in isolation or this region in the context of an Fc polypeptide,for example an antibody. The Fc may be a native sequence Fc or a variantFc. Replacements of amino acid residues in the Fc portion to alterantibody effector function are known in the art (see, e.g., Winter etal., U.S. Pat. Nos. 5,648,260 and 5,624,821). One suitable Fc, describedin PCT application WO 93/10151 (hereby incorporated by reference), is asingle chain polypeptide extending from the N-terminal hinge region tothe native C-terminus of the Fc region of a human IgG1 antibody. Anotheruseful Fc polypeptide is the Fc mutein described in U.S. Pat. No.5,457,035 and in Baum et al., 1994, EMBO J. 13:3992-4001. The amino acidsequence of this mutein is identical to that of the native Fc sequencepresented in WO 93/10151, except that amino acid 19 has been changedfrom Leu to Ala, amino acid 20 has been changed from Leu to Glu, andamino acid 22 has been changed from Gly to Ala. The mutein exhibitsreduced affinity for Fc receptors.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the Fc.gamma.RI,Fc.gamma.RII, and Fc.gamma.RIII subclasses, including allelic variantsand alternatively spliced forms of these receptors. Fc.gamma.RIIreceptors include Fc.gamma.RIIA (an “activating receptor”) andFc.gamma.RIIB (an “inhibiting receptor”), which have similar amino acidsequences that differ primarily in the cytoplasmic domains thereof.Activating receptor Fc.gamma.RIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor Fc.gamma.RIIB contains an immunoreceptortyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (seereview M. in Daeron, Annu. Rev. Immunol 15:203-234 (1997)). FcRs arereviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capelet al. Immunomethods 4:25-34 (1994); and de Haas et al, J. Lab. Clin;Med. 126:330-41 (1995). Other FcRs, including those to be identified inthe future, are encompassed by the term “FcR” herein. The term alsoincludes the neonatal receptor, FcRn, which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), and regulateshomeostasis of immunoglobulins.

“Fv” is also the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the VH-VL dimer. Collectively,the six hypervariable regions confer antigen-binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three hypervariable regions specific for an antigen) hasthe ability to recognize and bind antigen, although at a lower affinitythan the entire binding site.

As used herein, a humanized antibody that comprises a heavy or lightchain variable “framework region” from a particular human germlineimmunoglobulin sequence may contain amino acid differences as comparedto the heavy or light chain variable framework region of the particulargermline sequence, due to, for example, naturally-occurring somaticmutations or intentional introduction of site-directed mutation.However, a selected humanized antibody typically is at least 90%identical in amino acid sequence of the heavy or light chain variableframework region to an amino acid sequence encoded by the heavy or lightchain variable framework region of a human germline immunoglobulin geneand contains amino acid residues that identify the humanized antibody asbeing derived from human when compared to the germline immunoglobulinamino acid sequences of other species (e.g., murine germline sequences).In certain cases, a humanized antibody may be preferably at least 90%,more preferably at least 95%, more preferably at least 96%, mostpreferably at least 97%, in particular at least 98%, most particular atleast 99%, identical in amino acid sequence of the heavy or light chainvariable framework region to the amino acid sequence of the heavy orlight chain variable framework region encoded by the germlineimmunoglobulin gene. Typically, the heavy or light chain variableframework region of a humanized antibody derived from a particular humangermline sequence will display no more than 11 amino acid, preferably nomore than 5, or even more preferably no more than 4, 3, 2, or 1differences from the amino acid sequence of the heavy or light chainvariable framework region encoded by the human germline immunoglobulingene.

“Human effector cells” are leukocytes that express one or more FcRs andperform effector functions. Preferably, the cells express at leastFc.gamma.RIII and perform ADCC effector function. Examples of humanleukocytes which mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells andneutrophils; with PBMCs and NK cells being preferred. The effector cellsmay be isolated from a native source thereof, e.g. from blood or PBMCsas described herein.

“Human germline sequences” are found naturally in the human population.A combination of those germline genes generates antibody diversity.Germline antibody sequences for the light chain of the antibody comefrom conserved human germline kappa or lambda v-genes and j-genes.Similarly the heavy chain sequences come from germline v-, d- andj-genes (LeFranc, M-P, and LeFranc, G, “The Immunoglobulin Facts Book”Academic Press, 2001). Publicly available, well-known, databases existfor all known germline sequences.

The term “hinge” or “hinge region” or “antibody hinge region” hereinincludes the flexible polypeptide comprising the amino acids between thefirst and second constant domains of an antibody or tau-binding fragmentthereof. The “hinge region” as referred to herein is a sequence regionof 6-62 amino acids in length, only present in IgA, IgD and IgG, whichencompasses the cysteine residues that bridge the two heavy chains.Structurally, in one embodiment, the IgG CH1 domain ends at EU position220, and the IgG CH2 domain begins at residue EU position 237. Thus, forIgG the antibody hinge is herein defined, in one embodiment, to includepositions 221 (D221 in IgG1) to 231 (A231 in IgG1), wherein thenumbering is according to the EU numbering system.

The term “humanized antibody” refers to antibodies in which theframework regions (FR) and/or the complementarity determining regions(CDR) have been modified to comprise the CDR of an immunoglobulin ofdifferent specificity (mouse) as compared to that of the parentimmunoglobulin (human). In one embodiment, a murine CDR is grafted intothe framework region of a human antibody to prepare the “humanizedantibody.” In another embodiment, human frameworks are “grafted” orspliced into mouse antibodies, preserving the CDRs of the mouse antibodyand replacing its frameworks with frameworks of human origin. Graftingand splicing can be done by various recombinant DNA technologies,including PCR and mutagenesis. Various humanization methods exist in theart (e.g., CDR grafting, reshaping, transgenic animals, combinatoriallibraries). See, e.g., Riechmann, L., et al., Nature 332 (1988) 323-327;Neuberger, M. S., et al., Nature 314 (1985) 268-270; Sastry L,Alting-Mess M, Huse W D, Short J M, Sorge J A, Hay B N, Janda K D,Benkovic S J, Lerner R A (1989) Cloning of the immunological repertoirein for generation of monoclonal catalytic antibodies: construction of aheavy chain variable region-specific cDNA library. Proc Natl Acad SciUSA 86, 5728-5732; and Huse W D, Sastry S, Iverson S A, Kang A S,Alting-Mees M, Burton D R, Benkovic S J, Lerner R A (1989) Generation ofa large combinatorial library of the immunoglobulin repertoire in phagelambda. Science 246, 1275-1281. Humanized antibodies result fromgenetically engineering another antibody to make it more human-likewhile retaining its original antigen-binding properties. Presta, L. G.Engineering of therapeutic antibodies to minimize immunogenicity andoptimize function. Advanced Drug Delivery Reviews, Volume 58, Issues5-6: 640-656 (2006). The choice of human variable domains, both lightand heavy, to be used in making the humanized antibodies is veryimportant to reduce antigenicity. According to the so-called “best-fit”method, the sequence of the variable domain of a rodent antibody isscreened against a library of known human variable-domain sequences or alibrary of human germline sequences. The human sequence that is closestto that of the rodent can then be accepted as the human framework regionfor the humanized antibody (Sims et al., J. Immunol. 1993; 151:2296 etseq.; Chothia et al, Chothia and Lesk, J. Mol. Biol. 1987; 196:901-917).Another method uses a particular framework region derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., PNAS USA, 1992; 89:4285et seq.; Presta et al., J Immunol 1993; 151:2623 et seq.). Other methodsdesigned to reduce the immunogenicity of the antibody molecule in ahuman patient include veneered antibodies (see, e.g., U.S. Pat. No.6,797,492 and U.S. patent application publications 20020034765 and20040253645) and antibodies that have been modified by T-cell epitopeanalysis and removal (see, e.g., U.S. patent application publications20030153043 and U.S. Pat. No. 5,712,120).

Particularly preferred CDRs of the humanized antibodies described hereincorrespond to the CDR sequences of the mouse monoclonal DC8E8 antibody,namely SEQ ID NOs.1-6. A copy of the humanized antibodies andtau-binding fragments thereof described herein (e.g. an immunoglobulinwith the same heavy or light chain variable region as those describedherein), made by recombinant methods or any other methods (except anaturally existing antibody), is also a humanized antibody ortau-binding fragment thereof within the scope of the term.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody that are responsible for antigen-binding.In one embodiment, according to Kabat, the hypervariable regiongenerally comprises amino acid residues from a “complementaritydetermining region” or “CDR” (e.g. residues 24-34 (L1), 50-56 (L2) and89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2)and 95-102 (H3) in the heavy chain variable domain; Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (e.g. residues 26-32 (L1),50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32(H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain;Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

“Framework Region” or “FR” residues are those variable domain residuesother than the hypervariable region residues as herein defined. In theIMGT unique numbering system, the conserved amino acids always have thesame position, for instance cysteine 23 (1st-CYS), tryptophan 41(CONSERVED-TRP), hydrophobic amino acid 89, cysteine 104 (2nd-CYS),phenylalanine or tryptophan 118 (J-PHE or J-TRP). See, e.g., LefrancM.-P., Immunology Today 18, 509 (1997); Lefranc M.-P., The Immunologist,7, 132-136 (1999); Lefranc, M.-P., Pommié, C., Ruiz, M., Giudicelli, V.,Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, Dev. Comp.Immunol., 27, 55-77 (2003). In another embodiment, the IMGT uniquenumbering provides a standardized delimitation of the framework regions(FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104and FR4-IMGT: 118 to 128) and of the complementarity determiningregions: CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to117. As gaps represent unoccupied positions, the CDR-IMGT lengths (shownbetween brackets and separated by dots, e.g. [8.8.13]) become importantinformation. The IMGT unique numbering is used in 2D graphicalrepresentations, designated as IMGT Colliers de Perles. See, e.g., Ruiz,M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002); Kaas, Q. andLefranc, M.-P., Current Bioinformatics, 2, 21-30 (2007). It is also usedfor representing 3D structures. See, e.g., IMGT/3Dstructure-DB Kaas, Q.,Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data.Nucl. Acids. Res., 32, D208-D210 (2004). Framework or FR residues arethose variable domain residues other than and bracketing thehypervariable regions.

Using the Kabat numbering system, the actual linear amino acid sequencemay contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The term “immunogenicity” or immunogenic, as used herein, refers to anantibody response to administration of a therapeutic drug. Theimmunogenicity toward the antibodies and tau-binding fragments providedherein is obtained using quantitative and qualitative assays fordetection of antibodies against said therapeutic proteins, polypeptidesand peptides in biological fluids. Such assays include, but are notlimited to, Radioimmunoassay (RIA), Enzyme-linked immunosorbent assay(ELISA), luminescent immunoassay (LIA), and fluorescent immunoassay(FIA). Analysis of such immunogenicity involves comparing the antibodyresponse upon administration of antibodies and tau-binding fragmentsprovided herein to the antibody response upon administration of acontrol therapeutic protein, polypeptide or peptide or of the deliveryvehicle or delivery buffer.

The “light chains” of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (κ) andlambda (λ), based on the amino acid sequences of their constant domains.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (VH) followedby a number of constant domains. Each light chain has a variable domainat one end (VL) and a constant domain at its other end. The constantdomain of the light chain is aligned with the first constant domain ofthe heavy chain, and the light-chain variable domain is aligned with thevariable domain of the heavy chain. Particular amino acid residues arebelieved to form an interface between the light chain and heavy chainvariable domains.

The term “nucleic acid” as used herein, is intended to include DNAmolecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The “percentage identity” between two sequences of nucleic acids oramino acids means the percentage of identical nucleotides or amino acidresidues between the two sequences to be compared, obtained afteroptimal alignment, this percentage being purely statistical and thedifferences between the two sequences being distributed randomly alongtheir length. The comparison of two nucleic acid or amino acid sequencesis traditionally carried out by comparing the sequences after havingoptimally aligned them, said comparison being able to be conducted bysegment or by using an “alignment window”. Optimal alignment of thesequences for comparison can be carried out, in addition to comparisonby hand, by means of the local homology algorithm of Smith and Waterman(1981) [Ad. App. Math. 2:482], by means of the local homology algorithmof Neddleman and Wunsch (1970) [J. Mol. Biol. 48:443], by means of thesimilarity search method of Pearson and Lipman (1988) [Proc. Natl. Acad.Sci. USA 85:2444] or by means of computer software using thesealgorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis., or by the comparison software BLAST NR or BLAST P). For example,the identity of a sequence can exist over a region that is at leastabout 75-100 sequential units in length, over a region that is about 50sequential units in length, or, where not specified, across the entiresequence of a polypeptide sequence.

The percentage identity between two nucleic acid or amino acid sequencesis determined by comparing the two optimally-aligned sequences in whichthe nucleic acid or amino acid sequence to compare can have additions ordeletions compared to the reference sequence for optimal alignmentbetween the two sequences. Percentage identity is calculated bydetermining the number of positions at which the amino acid nucleotideor residue is identical between the two sequences, for example betweenthe two complete sequences, dividing the number of identical positionsby the total number of positions in the alignment window and multiplyingthe result by 100 to obtain the percentage identity between the twosequences. In one embodiment, percentage sequence identities betweenantibodies are determined with antibody sequences maximally aligned bythe Kabat numbering convention. After alignment, if a subject antibodyregion (e.g., the entire mature variable region of a heavy or lightchain) is being compared with the same region of a reference antibody,the percentage sequence identity between the subject and referenceantibody regions is the number of positions occupied by the same aminoacid in both the subject and reference antibody region divided by thetotal number of aligned positions of the two regions, with gaps notcounted, multiplied by 100 to convert to percentage.

For example, the BLAST program, “BLAST 2 sequences” (Tatusova et al.,“Blast 2 sequences—a new tool for comparing protein and nucleotidesequences”, FEMS Microbiol., 1999, Lett. 174:247-250) available on thesite http://www.ncbi.nlm.nih.gov/gorf/bl2.html, can be used with thedefault parameters (notably for the parameters “open gap penalty”: 5,and “extension gap penalty”: 2; the selected matrix being for examplethe “BLOSUM 62” matrix proposed by the program); the percentage identitybetween the two sequences to compare is calculated directly by theprogram.

For the amino acid sequence exhibiting at least 80%, for example 85%,90%, 95% and 98% identity with a reference amino acid sequence,preferred examples include those containing the reference sequence,certain modifications, notably a deletion, addition or substitution ofat least one amino acid, truncation or extension. In the case ofsubstitution of one or more consecutive or non-consecutive amino acids,substitutions are preferred in which the substituted amino acids arereplaced by “equivalent” amino acids. Here, the expression “equivalentamino acids” is meant to indicate any amino acids likely to besubstituted for one of the structural amino acids without howevermodifying the biological activities of the corresponding antibodies andof those specific examples defined below.

Equivalent amino acids can be determined either on their structuralhomology with the amino acids for which they are substituted or on theresults of comparative tests of biological activity between the variousantibodies likely to be generated. As a non-limiting example, the table1 below summarizes the possible substitutions likely to be carried outwithout resulting in a significant modification of the biologicalactivity of the corresponding modified antibody; inverse substitutionsare naturally possible under the same conditions.

TABLE 1 Original residue Substitution(s) Ala (A) Val, Gly, Pro Arg (R)Lys, His Asn (N) Gln Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) AspGly (G) Ala His (H) Arg Ile (I) Leu Leu (L) Ile, Val, Met Lys (K) ArgMet (M) Leu Phe (F) Tyr Pro (P) Ala Ser (S) Thr, Cys Thr (T) Ser Trp (W)Tyr Tyr (Y) Phe, Trp Val (V) Leu, Aa

In the context of this disclosure, the terms “pathological tau” and“disease tau” include pathological tau conformers and structures andencompasses all of the following: Tau Type IA, IB, IIA, and IIB(described in detail in WO2004/007547 A2), misordered, misdisordered tau(monomer, dimer, trimer, oligomer), misdisordered soluble tau,sarkosyl-insoluble tau, extracellular tau deposits, tau aggregates,paired helical filaments, neurofibrillary pathology, includingneurofibrillary lesions, tangles, threads, fibrils, axonal spheriods,highly phosphorylated forms of truncated tau and of full-length tau, orany other form of tau associated with AD or another tauopathy that isdetectable by the antibodies and/or tau-binding fragments describedherein.

Tau 151-391/4R (also referred to as tauΔ(1-150:392-441)/4R) represents aform of pathological tau.

The term “pharmaceutically acceptable” means biologically orpharmacologically compatible for in vivo use in animals or humans, andpreferably means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

The term “another tauopathy” encompasses all neurological diseases thatare accompanied by the appearance of abnormal forms of microtubuleassociated protein tau in the brains of patients. The term includes, butis not limited to, the following diseases: Alzheimer's disease,Gerstmann-Sträussler-Scheinker disease, British dementia, Danishdementia, Pick's disease, Progressive supranuclear palsy, Corticobasaldegeneration, Argyrophilic grain disease, Guam Parkinsonism-dementiacomplex, Tangle-only dementia, White matter tauopathy with globularglial inclusions, Frontotemporal dementia (e.g., FTDP-17), andParkinsonism linked to chromosome 17. See, e.g., Goedert M, Clavaguera Fand Tolnay M. The propagation of prion-like protein inclusions inneurodegenerative diseases. Trends Neural. Sci. In one embodiment, oneor more of those abnormal forms of tau is recognized by one of theantibodies or binding fragments described herein in at least one assay.In some embodiments, the assay is IHC. In other embodiments, the assayis ELISA.

As used herein “specifically binds” in reference to an antibody meansthat the antibody binds to its target antigen or epitope with greateraffinity that it does to a structurally different antigen(s) or epitope.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Example 1 and Jonsson, U., et al. (1993) Ann.Biol. Clin. 51:19-26; Jonsson, U., et al. (1991) Biotechniques11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; andJohnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFvsee Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term “treatment” as used herein, is defined as the application oradministration of a therapeutic agent to a subject, who has a disease, asymptom of disease or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease, the symptoms of disease or thepredisposition toward disease. Moreover, as long as the compositions ofthe disclosure either alone or in combination with another therapeuticagent cure, heal, alleviate, relieve, alter, remedy, ameliorate, improveor affect at least one symptom of Alzheimer's Disease or anothertauopathy being treated as compared to that symptom in the absence ofuse of the humanized anti-tau antibody or tau-binding fragment thereofcomposition, the result should be considered a treatment of theunderlying disorder regardless of whether all the symptoms of thedisorder are cured, healed, alleviated, relieved, altered, remedied,ameliorated, improved or affected or not.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework regions (FRs). The variabledomains of native heavy and light chains each comprise four FRs, largelyadopting a beta-sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the beta-sheet structure. The hypervariable regions (HVR) in each chainare held together in close proximity by the FRs and, with thehypervariable regions from the other chain, contribute to the formationof the antigen-binding site of antibodies (see Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody dependent cellular cytotoxicity (ADCC).

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The present disclosure may be understood more readily by reference tothe following detailed description of specific embodiments includedherein. Although the present disclosure has been described withreference to specific details of certain embodiments thereof, it is notintended that such details should be regarded as limitations upon thescope of the disclosure.

Humanized Antibodies that Bind to Disease Tau

Provided herein are the first humanized antibodies against humandisease/pathological tau that are capable of recognizing four differentregions of tau in a conformation-dependent manner. More particularly,the humanized antibodies described herein are capable of binding atleast one, two, three, or four of QT1-QT4 in such manner that theydiscriminate between wild-type/normal tau and conformations of tau thatare associated with AD (disease tau). In some embodiments, two, three,or four of the QT1-QT4 may be simultaneously occupied by theseantibodies. In other words, the antibodies and tau-binding fragmentsdescribed herein have as their “epitope” any one or more of thefollowing four tau sites, some or all of which are conformationalepitopes: 268-HQPGGG-273 (SEQ ID NO: 148) (within 1^(st) repeat domainof tau protein), 299-HVPGGG-304 (SEQ ID NO: 149) (within 2^(nd) repeatdomain of tau protein), 330-HKPGGG-335 (SEQ ID NO: 150) (within 3^(rd)repeat domain of tau protein), and 362-HVPGGG-367 (SEQ ID NO: 149)(within 4^(th) repeat domain of tau protein). These epitopes are alsoreferred herein as QT1, QT2, QT3, and QT4, respectively. DC8E8 binds toall of QT1-QT4; in fact, the epitope of DC8E8 is HXPGGG, wherein X isany amino acid.

In one embodiment, the antibodies and tau-binding fragments haveaffinities for Tau 151-139/4R that are at least 80% as good if notbetter than that of the parent mouse monoclonal antibody DC8E8. In oneembodiment, the antibodies and tau-binding fragments retain specificityfor the epitopes recognized by the mouse DC8E8 antibody. In oneembodiment, these antibodies have one or more advantageous biochemicalproperties (e.g., human constant regions and thus reducedimmunogenecity, high expression levels, high solubility, lack ofsignificant protein aggregation upon purification, high stability) thatrender them optimal for use in the clinic for treatment of AD andrelated tauopathies in humans.

Humanization of DC8E8 was empirically optimized by manipulation offramework residues, The initial humanized version RHA/RKA (AX001)exhibited a 10-fold decrease in binding affinity for tau, relative tothe chimeric DC8E8 construct. These data implied that one or moreframework amino acid residues are important in order to humanize DC8E8with little resultant loss in binding activity. In contrast, a singleframework back-mutation was sufficient to restore binding affinity inthe RHD (AX004) humanized antibody. This superiority was unexpected.Other single point back-mutations did not have the same unexpectedadvantage in binding affinity. See, for example, RHF/RKA (AX006) andRHG/RKA (AX007) constructs. And the next best antibody carried 10back-mutations. See AX002. Of all back-mutation combinations tested,AX004 proved to have the best binding affinity for tau (in the assaydescribed herein), despite having a single back-mutation, and unlikeother single back-mutation constructs. Other humanized constructs ofimproved affinity include, for example, AX002, AX005, AX037 and AX014,AX016, AX017, AX038, in RKA and RKB light chain versions, respectively.

Also provided are tau-binding fragments (e.g. antibody portions) of thehumanized antibodies described herein. These fragments are also capableof binding to at least one, two, three, or four of QT1-QT4 (definedabove) in such manner that they discriminate between wild-type/normaltau and conformations of tau that are associated with AD. In someembodiments, all four of the QT1-QT4 epitopes may be occupied by four ofthe antibodies or tau-binding fragments described herein. In someembodiments, the fragments or portions bind to tau with the sameaffinity and properties of the mouse monoclonal antibody DC8E8. Forexample, antibody fragments or portions capable of binding to one ormore of QT1-QT4, include, but are not limited to Fab (e.g., by papaindigestion), Fd, Fab′ (e.g., by pepsin digestion and partial reduction)and F(ab′)₂ (e.g., by pepsin digestion), facb (e.g., by plasmindigestion), pFc′ (e.g., by pepsin or plasmin digestion), Fd (e.g., bypepsin digestion, partial reduction and re-aggregation), Fv or scFv(e.g., by molecular biology techniques) fragments, are provided by thepresent invention. See also, William E. Paul (ed.) FundamentalImmunology, 6^(th) Edition, Lippincott Williams & Wilkins, NY, N.Y.(2008), incorporated herein by reference in its entirety. Any otherfragments whose half-life has been increased by conjugation to othermolecules, including PEGylated fragments, are also within the scope ofthis invention. Certain fragments can be produced by enzymatic cleavage,synthetic or recombinant techniques, as routinely known in the art, oras provided herein. Antibodies can also be produced in a variety oftruncated forms using antibody genes in which one or more stop codonshave been introduced upstream of the natural stop site. For example, acombination gene encoding an F(ab′)₂ heavy chain portion can be designedto include DNA sequences encoding the CH1 domain and/or hinge region ofthe heavy chain. The various portions of antibodies can be joinedtogether chemically by conventional techniques, or can be prepared as acontiguous protein using routine genetic engineering techniques.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes”.In one embodiment, there are six major classes of intact antibodies:IgA, IgD, IgE, IgG, IgY, and IgM, and several of these may be furtherdivided into “subclasses” (isotypes), e.g., IgG1 IgG2, IgG3, IgG4, IgA,and IgA2. The heavy-chain constant domains that correspond to thedifferent classes of antibodies are called alpha (α), delta (δ), epsilon(ε), gamma (γ), and mu (μ), respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known. In one embodiment, the antibodies described herein haveconstant regions of any of the existing immunogenic isotypes. Forexample, the constant regions can be of the lambda or kappa region andthe gamma-1, gamma-2, gamma-3, or gamma 4 regions. Constant regions ofmixed isotypes are also within the scope of this disclosure (e.g., IgG1mixed with IgG4). It has been perceived that some isotypes are superiorover the others. Bruggemann M, Williams G T, Bindon C I, Clark M R,Walker M R, Jefferis R, Waldmann H, Neuberger M S (1987) Comparison ofthe effector functions of human immunoglobulins using a matched set ofchimeric antibodies. J. Exp. Med. 166, 1351-1361; Bindon C I, Hale G,Bruggemann M, Waldmann H (1988) Human monoclonal IgG isotypes differincomplement activating function at the level of C4 as well as C1 q. J.Exp. Med. 168, 127-42; Shaw D R, Khazaeli M B, LoBuglio A F (1988)Mouse/human chimeric antibodies to a tumor associated antigen: biologicactivity of the four human IgG subclasses. J. Natl. Cancer Inst. 80,1553-9; Steplewski Z, Sun L K, Shearman C W, Ghrayeb J, Daddona P,Koprowski, H (1988) Biological activity of human-mouse IgG1, IgG2, IgG3,and IgG4chimeric monoclonal antibodies with antitumor specificity. Proc.Natl. Acad. Sci. USA 85, 4852-6. In one set of experiments, described inthese references, the human isotypes IgM, IgG1, IgG2, IgG3 (twoallotypes), IgG4, IgA and IgE were compared for autologous complementmediated lysis as well as for antibody dependent cell mediatedcytotoxicity (ADCC). For complement mediated lysis, human IgM and IgG1proved to be the most effective with the two allotypes of IgG3 beingnext best. IgG2 gave poor lysis while the other isotypes seemed to giveno lysis. The results for ADCC were that IgG1 was again very effectivefollowed by either IgG2, IgG3, or IgG4, depending on the assay (e.g.,cell type). The other isotypes, including IgM, were ineffective. Butantibodies have many other activities (e.g., promoting opsonization bymacrophages), and ultimately it is difficult or even not possible topredict which isotype is going to have the best set of properties forits desired use.

Another factor influencing the immunogenicity of a humanized antibody isthe existence of polymorphic determinants in the constant region. Thesedifferences can be minimized for reduced antigenicity. There are 18allotypes of human IgG observed with reasonable frequencies in thepopulation: IgG1 has 4; IgG2 has 1; IgG3 has 13; IgG4 has 0. WHO (1976)Review of the notation for the allotypic and related markers of humanimmunoglobulins. Eur. J. Immunol. 6, 599-601. In addition there arethree allotypes of human k light chains. Some allotypes are present insome individuals and not in others. Some are predominantly expressedamongst the Japanese population.

Many methods for humanizing non-human antibodies have been described inthe art. In one embodiment, a humanized antibody has one or more aminoacid residues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain. Inone embodiment, humanization can be essentially performed following themethod of Winter and co-workers (Jones et al, Nature, 321:522-525(1986); Riechmann et al, Nature, 332:323-327 (1988); Verhoeyen et al.,Science, 239:1534-1536 (1988)), by substituting hypervariable regionsequences for the corresponding sequences of a human antibody.Accordingly, such “humanized” antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567) wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In some embodiments, humanized antibodies are humanantibodies in which some hypervariable region residues and possibly someFR residues are substituted by residues from analogous sites in rodentantibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is important to reduce antigenicity.According to the so-called “best-fit” method, the sequence of thevariable domain of a rodent antibody is screened against the entirelibrary of known human variable-domain sequences. The human sequence(entire variable region or just the frameworks) that is closest to thatof the rodent is then accepted as the human framework region (FR) forthe humanized antibody (Sims et al, J. Immunol., 151:2296 (1993);Chothia et al, J. Mol. Biol., 196:901 (1987)). Another method uses aparticular framework region derived from the consensus sequence of allhuman antibodies of a particular subgroup of light or heavy chains. Thesame framework may be used for several different humanized antibodies(Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta etal., J. Immunol, 151:2623 (1993)).

In some embodiments, it is important that antibodies be humanized withretention of high affinity for the antigen and other favorablebiological properties of the parent (e.g., mouse antibody). To achievethis goal, according to one embodiment, humanized antibodies areprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from therecipient and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the hypervariable region residues are directly andmost substantially involved in influencing antigen binding.

TABLE 2 VARIABLE POLYPEPTIDE SEQ ID NO. REGION ONLY SEQ ID NO. CDR-H1 1HEAVY DC8E8 7 CDR-H2 2 LIGHT DC8E8 8 CDR-H3 3 HEAVY  9 (identical to 7)CHIMERIC CDR-L1 4 LIGHT 10 (identical to 8) CHIMERIC CDR-L2 5 HEAVYDC8E8 11 (identical to 7) OPTIMIZED CDR-L3 6 LIGHT DCBE8 12 (identicalto 8) OPTIMIZED

The previous table 2 provides the SEQ ID NOs. for some of the amino acidsequences described herein. In another embodiment, a humanized antibodyor tau-binding fragment, as described herein, comprises SEQ ID NOs. 1,2, 3, as the heavy chain CDRs 1, 2, and 3, respectively, and SEQ ID Nos.4, 5, 6, as the light chain CDRs 1, 2, and 3, respectively. In someembodiments, this antibody or fragment comprises at least one CDR, asdefined according to Kabat, whose sequence has at least 80%, preferableat least 85%, 90%, 95%, and 98% identity after optimal alignment with aCDR of any one of SEQ ID NOs. 1 to 6.

In one embodiment, the antibody (AX001) or tau-binding fragment thereofcomprises heavy chain variable region RHA (SEQ ID NO. 13) and lightchain variable region RKA (SEQ ID NO. 26). This antibody is providedwith either IgG1 (AX001-IgG1) or IgG4 (AXON001-IgG4) constant regions.

In one embodiment, the antibody (AX002) or tau-binding fragment thereofcomprises heavy chain variable region RHB (SEQ ID NO. 14) and lightchain variable region RKA (SEQ ID NO. 26). This antibody is providedwith either IgG1 (AX002-IgG1) or IgG4 (AXON002-IgG4) constant regions.

In one embodiment, the antibody (AX003) or tau-binding fragment thereofcomprises heavy chain variable region RHC (SEQ ID NO. 15) and lightchain variable region RKA (SEQ ID NO. 26). This antibody is providedwith either IgG1 (AX003-IgG1) or IgG4 (AXON003-IgG4) constant regions.

In one embodiment, the antibody (AX004) or tau-binding fragment thereofcomprises heavy chain variable region RHD (SEQ ID NO. 16) and lightchain variable region RKA (SEQ ID NO. 26). This antibody is providedwith either IgG1 (AX004-IgG1) or IgG4 (AXON004-IgG4) constant regions.

In one embodiment, the antibody (AX005) or tau-binding fragment thereofcomprises heavy chain variable region RHE (SEQ ID NO. 17) and lightchain variable region RKA (SEQ ID NO. 26). This antibody is providedwith either IgG1 (AX005-IgG1) or IgG4 (AXON005-IgG4) constant regions.

In one embodiment, the antibody (AX006) or tau-binding fragment thereofcomprises heavy chain variable region RHF (SEQ ID NO. 18) and lightchain variable region RKA (SEQ ID NO. 27). This antibody is providedwith either IgG1 (AX006-IgG1) or IgG4 (AXON006-IgG4) constant regions.

In one embodiment, the antibody (AX007) or tau-binding fragment thereofcomprises heavy chain variable region RHG (SEQ ID NO. 19) and lightchain variable region RKA (SEQ ID NO. 26). This antibody is providedwith either IgG1 (AX007-IgG1) or IgG4 (AXON007-IgG4) constant regions.

In one embodiment, the antibody (AX008) or tau-binding fragment thereofcomprises heavy chain variable region RHH (SEQ ID NO. 20) and lightchain variable region RKA (SEQ ID NO. 26). This antibody is providedwith either IgG1 (AX008-IgG1) or IgG4 (AXON008-IgG4) constant regions.

In one embodiment, the antibody (AX009) or tau-binding fragment thereofcomprises heavy chain variable region RHI (SEQ ID NO. 21) and lightchain variable region RKA, (SEQ ID NO. 26). This antibody is providedwith either IgG1 (AX009-IgG1) or IgG4 (AXON009-IgG4) constant regions.

In one embodiment, the antibody (AX010) or tau-binding fragment thereofcomprises heavy chain variable region RHJ (SEQ ID NO. 22) and lightchain variable region RKA (SEQ ID NO. 26). This antibody is providedwith either IgG1 (AX010-IgG1) or IgG4 (AXON010-IgG4) constant regions.

In one embodiment, the antibody (AX011) or tau-binding fragment thereofcomprises heavy chain variable region RHK (SEQ ID NO. 23) and lightchain variable region RKA (SEQ ID NO. 26). This antibody is providedwith either IgG1 (AX011-IgG1) or IgG4 (AXON011-IgG4) constant regions.

In one embodiment, the antibody (AX012) or tau-binding fragment thereofcomprises heavy chain variable region RHL (SEQ ID NO. 24) and lightchain variable region RKA (SEQ ID NO. 26). This antibody is providedwith either IgG1 (AX012-IgG1) or IgG4 (AXON012-IgG4) constant regions.

In one embodiment, the antibody (AX013) or tau-binding fragment thereofcomprises heavy chain variable region RHA (SEQ ID NO. 1) and light chainvariable region RKB (SEQ ID NO. 27). This antibody is provided witheither IgG1 (AX013-IgG1) or IgG4 (AXON013-IgG4) constant regions.

In one embodiment, the antibody (AX014) or tau-binding fragment thereofcomprises heavy chain variable region RHB (SEQ ID NO. 2) and light chainvariable region RKB (SEQ ID NO. 27). This antibody is provided witheither IgG1 (AX014-IgG1) or IgG4 (AXON014-IgG4) constant regions.

In one embodiment, the antibody (AX015) or tau-binding fragment thereofcomprises heavy chain variable region RHC (SEQ ID NO. 15) and lightchain variable region RKB (SEQ ID NO. 27). This antibody is providedwith either IgG1 (AX015-IgG1) or IgG4 (AXON015-IgG4) constant regions.

In one embodiment, the antibody (AX016) or tau-binding fragment thereofcomprises heavy chain variable region RHD (SEQ ID NO. 16) and lightchain variable region RKB (SEQ ID NO. 27). This antibody is providedwith either IgG1 (AX016-IgG1) or IgG4 (AXON016-IgG4) constant regions.

In one embodiment, the antibody (AX017) or tau-binding fragment thereofcomprises heavy chain variable region RHE (SEQ ID NO. 17) and lightchain variable region RKB (SEQ ID NO. 27). This antibody is providedwith either IgG1 (AX017-IgG1) or IgG4 (AXON017-IgG4) constant regions.

In one embodiment, the antibody (AX018) or tau-binding fragment thereofcomprises heavy chain variable region RHF (SEQ ID NO. 18) and lightchain variable region RKB (SEQ ID NO. 27). This antibody is providedwith either IgG1 (AX018-IgG1) or IgG4 (AXON018-IgG4) constant regions.

In one embodiment, the antibody (AX019) or tau-binding fragment thereofcomprises heavy chain variable region RHG (SEQ ID NO. 19) and lightchain variable region RKA (SEQ ID NO. 27). This antibody is providedwith either IgG1 (AX019-IgG1) or IgG4 (AXON019-IgG4) constant regions.

In one embodiment, the antibody (AX020) or tau-binding fragment thereofcomprises heavy chain variable region RHH (SEQ ID NO. 20) and lightchain variable region RKB (SEQ ID NO. 27). This antibody is providedwith either IgG1 (AX020-IgG1) or IgG4 (AXON020-IgG4) constant regions.

In one embodiment, the antibody (AX021) or tau-binding fragment thereofcomprises heavy chain variable region RHI (SEQ ID NO. 21) and lightchain variable region RKB (SEQ ID NO. 27). This antibody is providedwith either IgG1 (AX021-IgG1) or IgG4 (AXON021-IgG4) constant regions.

In one embodiment, the antibody (AX022) or tau-binding fragment thereofcomprises heavy chain variable region RHJ (SEQ ID NO. 22) and lightchain variable region RKB (SEQ ID NO. 27). This antibody is providedwith either IgG1 (AX022-IgG1) or IgG4 (AXON022-IgG4) constant regions.

In one embodiment, the antibody (AX023) or tau-binding fragment thereofcomprises heavy chain variable region RHK (SEQ. ID NO. 23) and lightchain variable region RKB (SEQ ID NO. 27). This antibody is providedwith either IgG1 (AX023-IgG1) or IgG4 (AXON023-IgG4) constant regions.

In one embodiment, the antibody (AX024) or tau-binding fragment thereofcomprises heavy chain variable region RHL (SEQ ID NO. 24) and lightchain variable region RKB (SEQ ID NO. 27). This antibody is providedwith either IgG1 (AX024-IgG1) or IgG4 (AXON024-IgG4) constant regions.

In one embodiment, the antibody (AX025) or tau-binding fragment thereofcomprises heavy chain variable region RHA (SEQ ID NO. 13) and lightchain variable region VK (SEQ ID NO. 8)). This antibody is provided witheither IgG1 (AX025-IgG1) or IgG4 (AXON025-IgG4) constant regions.

In one embodiment, the antibody (AX026) or tau-binding fragment thereofcomprises heavy chain variable region RHB (SEQ ID NO. 14) and lightchain variable region VK (SEQ ID NO.8). This antibody is provided witheither IgG1 (AX025-IgG1) or IgG4 (AXON026-IgG4) constant regions.

In one embodiment, the antibody (AX027) or tau-binding fragment thereofcomprises heavy chain variable region RHC (SEQ ID NO. 15) and lightchain variable region VK (SEQ ID NO.8). This antibody is provided witheither IgG1 (AX027-IgG1) or IgG4 (AXON027-IgG4) constant regions.

In one embodiment, the antibody (AX028) or tau-binding fragment thereofcomprises heavy chain variable region RHD (SEQ ID NO. 16) and lightchain variable region VK (SEQ ID NO.8). This antibody is provided witheither IgG1 (AX028-IgG1) or IgG4 (AXON028-IgG4) constant regions.

In one embodiment, the antibody (AX029) or tau-binding fragment thereofcomprises heavy chain variable region RHE (SEQ ID NO. 17) and lightchain variable region VK (SEQ ID NO.8). This antibody is provided witheither IgG1 (AX029-IgG1) or IgG4 (AXON029-IgG4) constant regions.

In one embodiment, the antibody (AX030) or tau-binding fragment thereofcomprises heavy chain variable region RHF (SEQ ID NO. 18) and lightchain variable region VK (SEQ ID NO.8). This antibody is provided witheither IgG1 (AX030-IgG1) or IgG4 (AXON030-IgG4) constant regions.

In one embodiment, the antibody (AX031) or tau-binding fragment thereofcomprises heavy chain variable region RHG (SEQ ID NO. 19) and lightchain variable region VK (SEQ ID NO.8). This antibody is provided witheither IgG1 (AX031-IgG1) or IgG4 (AXON031-IgG4) constant regions.

In one embodiment, the antibody (AX032) or tau-binding fragment thereofcomprises heavy chain variable region RHH (SEQ ID NO. 20) and lightchain variable region VK (SEQ ID NO.8). This antibody is provided witheither IgG1 (AX032-IgG1) or IgG4 (AXON032-IgG4) constant regions.

In one embodiment, the antibody (AX033) or tau-binding fragment thereofcomprises heavy chain variable region RHI (SEQ ID NO. 21) and lightchain variable region VK (SEQ ID NO.8). This antibody is provided witheither IgG1 (AX033-IgG1) or IgG4 (AXON033-IgG4) constant regions.

In one embodiment, the antibody (AX034) or tau-binding fragment thereofcomprises heavy chain variable region RHJ (SEQ ID NO. 22) and lightchain variable region VK (SEQ ID NO.8). This antibody is provided witheither IgG1 (AX034-IgG1) or IgG4 (AXON034-IgG4) constant regions.

In one embodiment, the antibody (AX035) or tau-binding fragment thereofcomprises heavy chain variable region RHK (SEQ ID NO. 23) and lightchain variable region VK (SEQ ID NO.8). This antibody is provided witheither IgG1 (AX035-IgG1) or IgG4 (AXON035-IgG4) constant regions.

In one embodiment, the antibody (AX036) or tau-binding fragment thereofcomprises heavy chain variable region RHL (SEQ ID NO. 24) and lightchain variable region VK (SEQ ID NO.8). This antibody is provided witheither IgG1 (AX036-IgG1) or IgG4 (AXON036-IgG4) constant regions.

In one embodiment, the antibody (AX037) or tau-binding fragment thereofcomprises heavy chain variable region RHM (SEQ ID NO. 25) and lightchain variable region RKA (SEQ ID NO. 26). This antibody is providedwith either IgG1 (AX037-IgG1) or IgG4 (AXON037-IgG4) constant regions.

In one embodiment, the antibody (AX038) or tau-binding fragment thereofcomprises heavy chain variable region RHM (SEQ ID NO.) and light chainvariable region RKB (SEQ ID NO. 27). This antibody is provided witheither IgG1 (AX037-IgG1) or IgG4 (AXON037-IgG4) constant regions.

In another embodiment, the invention provides an antibody heavy chaincomprising a variable region chosen from any one of heavy chain variableregions RHA, RHB, RHC, RHD, RHE, RHF, RHG, RHH, RHI, RHJ, RHK, RHL, andRHM. Any of these variable regions can be linked to a constant region ofany human isotype, including constant regions with mixed isotypes.

In another embodiment, the invention provides an antibody light chaincomprising a variable region chosen from RKA and RKB. Any of thesevariable regions can be linked to a constant region of any humanisotype, including constant regions with mixed isotypes.

In another embodiment, the invention provides an antibody heavy chain ofany one of SEQ ID NO. 28-40 and 43-55.

In another embodiment, the invention provides an antibody light chainchosen from SEQ ID NO. 57-59.

The heavy and light chain variable regions of humanized antibodies canbe linked to at least a portion of a human constant region. As describedabove in the Definitions, the choice of constant region depends, inpart, whether antibody-dependent cell-mediated cytotoxicity, antibodydependent cellular phagocytosis and/or complement dependent cytotoxicityare desired. In one embodiment, human isotopes IgG1 and IgG3 havecomplement-dependent cytotoxicity and human isotypes IgG2 and IgG4 donot. In one embodiment, human IgG1 and IgG3 also induce stronger cellmediated effector functions than human IgG2 and IgG4. Light chainconstant regions can be lambda or kappa. An exemplary human light chainkappa constant region has the amino acid sequence of SEQ ID NO: 170. TheN-terminal arginine of SEQ ID NO:170 can be omitted, in which case lightchain kappa constant region has the amino acid sequence of SEQ IDNO:171. An exemplary human IgG1 heavy chain constant region has theamino acid sequence of SEQ ID NO:172. An exemplary human IgG4 heavychain constant region has the amino acid sequence of SEQ ID NO:173.Antibodies can be expressed as tetramers containing two light and twoheavy chains, as separate heavy chains, light chains, as Fab, Fab′,F(ab′)2, and Fv, or as single chain antibodies in which heavy and lightchain mature variable domains are linked through a spacer.

cDNA sequences encoding the constant regions of human antibodies areknown to one of ordinary skill in the art. In one embodiment, exemplarycDNA sequences available via, e.g., GenBank, each of which incorporatedby reference in its entirety, are as follows: Human IgG1 constant heavychain region: GenBank accession No.: J00228; Human IgG2 constant heavychain region: GenBank accession No.: J00230; Human IgG3 constant heavychain region: GenBank accession No.: X04646; Human IgG4 constant heavychain region: GenBank accession No.: K01316; and Human kappa light chainconstant region: GenBank accession No.: J00241. In one embodiment, theconstant region may further be modified according to known methods. Forexample, in an IgG4 constant region, residue S241 may be mutated to aproline (P) residue to allow complete disulphide bridge formation at thehinge (see, e.g., Angel et al., Mol. Immunol. 1993; 30:105-8).

For more clarity, the table 3 below summarizes the various amino acidsequences corresponding to the various variable regions of some of thehumanized antibodies described herein.

TABLE 3 HUMANIZED HUMANIZED VARIABLE HEAVY VARIABLE LIGHT REGION ONLYSEQ ID NO. REGION ONLY SEQ ID NO. RHA 13 RKA 26 RHB 14 RKB 27 RHC 15 RHD16 RHE 17 RHF 18 RHG 19 RHH 20 RHI 21 RHJ 22 RHK 23 RHL 24 RHM 25

Table 4 below summarizes the amino acid sequences corresponding to thevarious full length sequences of the mouse and humanized antibodiesdescribed herein.

TABLE 4 COMPLETE COMPLETE VARIABLE AND VARIABLE AND HEAVY CON- HEAVYCON- STANT IgG1 SEQ ID NO. STANT IgG4 SEQ ID NO. RHA 28 RHA 43 RHB 29RHB 44 RHC 30 RHC 45 RHD 31 RHD 46 RHE 32 RHE 47 RHF 33 RHF 48 RHG 34RHG 49 RHH 35 RHH 50 RHI 36 RHI 51 RHJ 37 RHJ 52 RHK 38 RHK 53 RHL 39RHL 54 RHM 40 RHM 55 cDC8E8 41 cDC8E8 56 Mouse DC8E8 42

The table 5 below summarizes the various amino acid sequencescorresponding to the various full length sequences of the light chainsof the humanized antibodies described herein.

TABLE 5 COMPLETE HUMANIZED VARIABLE LIGHT CONSTANT K SEQ ID NO. RKA 57RKB 58 cDC8E8 kappa 59

Also provided are chimeric antibodies and tau-binding fragments thereof.In one embodiment, the chimeric antibody or tau-binding fragment thereofcomprises heavy chain variable region DC8E8 VH (SEQ ID NO. 9) and lightchain variable region DC8E8 VK(SEQ ID NO. 10) together with a human IgG1constant region (SEQ ID NO. 172) for the heavy chain and a kappaconstant region (SEQ ID NO. 170) for the light chain. In anotherembodiment, the chimeric antibody or tau-binding fragment thereofcomprises heavy chain variable region DC8E8 VH (SEQ ID NO. 9) and lightchain variable region DC8E8 VK(SEQ ID NO. 10) together with a human IgG4constant region (SEQ ID NO. 173) for the heavy chain and a kappaconstant region (SEQ ID NO. 170) for the light chain.

In one embodiment, the humanized antibody or a tau-bindingfragment/portion thereof is made recombinantly. In another embodiment,the humanized antibody or a tau-binding fragment/portion thereof ismade, at least partially, by chemical synthesis. In one embodiment, thechimeric antibody or a tau-binding fragment/portion thereof is maderecombinantly. In another embodiment, the chimeric antibody or atau-binding fragment/portion thereof is made, at least partially, bychemical synthesis.

Table 6 summarizes the sequences for some of the otherhumanization-related molecules described herein.

TABLE 6 AMINO ACID NUCLEIC ACID MOLECULE SEQ ID NO. SEQ ID NO. Y15982Igkv8-21*01 60 72 L17135 Igkv8-28*02 61 73 Y15980 IGKV8-19*01 62 74AJ235948 IGKV8-30*01 63 75 AJ235947 IGKV8-28*01 64 76 X72449 65 77AC160990 Musmus IGHV1-81*01 66 78 AC160473 Musmus IGHV1-77*01 67 79AC160990 Musmus IGHV1-83*01 68 80 AC160473 Musmus IGHV1-75*01 69 81X02064 Musmus IGHV1-54*02 70 82 M65092 71 83

Amino acid sequence modification(s) of the antibodies described hereinare contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody.Amino acid sequence variants of the antibody are prepared by introducingappropriate nucleotide changes into the antibody nucleic acid, or bypeptide synthesis. Such modifications include, for example, deletionsfrom, and/or insertions into and/or substitutions of, residues withinthe amino acid sequences of the antibody. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe antibody, such as changing the number or position of glycosylationsites.

Also comprised by the present disclosure are antibodies and bindingfragments the sequence of which has been altered by introducing at leastone, particularly at least two, more particularly at least 3 or moreconservative substitutions into the sequences of SEQ ID NOs: 13-25 andSEQ ID NOs: 26 and 27 respectively, such that the antibody essentiallymaintains its full functionality.

Certain amino acids from the human mature variable region frameworkresidues can be selected for substitution based on their possibleinfluence on CDR conformation and/or binding to antigen, mediatinginteraction between heavy and light chains, interaction with theconstant region, being a site for desired or undesiredpost-translational modification, being an unusual residue for itsposition in a human variable region sequence and therefore potentiallyimmunogenic, among other reasons. One of ordinary skill in the art wouldknow how to pick certain amino acids for substitution and then assessthe result of such substitution. In many embodiments, positions forsubstitution within frameworks and amino acids to substitute areselected empirically.

Amino acid substitutions can also be made in the CDRs. One possiblevariation is to substitute certain residues in the CDRs of the mouseDC8E8 antibody with corresponding residues from human CDRs sequences,typically from the CDRs of the human acceptor sequences used indesigning the exemplified humanized antibodies. In some antibodies onlypart of the CDRs, namely the subset of CDR residues required forbinding, termed the SDRs, are needed to retain binding in a humanizedantibody. CDR residues not contacting antigen and not in the SDRs can beidentified based on previous studies (for example residues H60-H65 inCDR H2 are often not required), from regions of Kabat CDRs lying outsideChothia hypervariable loops (Chothia, J. Mol. Biol. 196:901, 1987), bymolecular modeling and/or empirically, or as described in Gonzales etal. Mol. Immunol. 41:863 (2004). In such humanized antibodies atpositions in which one or more donor CDR residues is absent or in whichan entire donor CDR is omitted, the amino acid occupying the positioncan be an amino acid occupying the corresponding position (by Kabatnumbering) in the acceptor antibody sequence. The number of suchsubstitutions of acceptor for donor amino acids in the CDRs to includereflects a balance of competing considerations. Such substitutions arepotentially advantageous in decreasing the number of mouse amino acidsin a humanized antibody and consequently decreasing potentialimmunogenicity. However, substitutions can also cause changes ofaffinity, and significant reductions in affinity are preferably avoided.Positions for substitution within CDRs and amino acids to substitute canalso be selected empirically.

In one embodiment, antibodies and tau-binding fragments thereof comprisea heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 ofSEQ ID NOs 1, 2, and 3, respectively, and being at least 90% identicalto the mature heavy chain of SEQ ID NO. 28-40 (complete RHA-RHM); and alight chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of SEQID NOs. 4, 5, and 6 respectively, and being at least 90% identical tothe mature light chain of SEQ ID NO. 57 (RKA) or SEQ ID NO. 58 (RKB). Insome embodiments, the mature heavy chain variable region is at least95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO. 28-40. Insome embodiments, the mature light chain variable region is at least95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO. 57or SEQID NO. 58.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells Science,244:1081-1085 (1989). Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includeantibody with an N-terminal methionyl residue or the antibody fused toanother therapeutic agent. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g. for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. In one embodiment, the sites ofgreatest interest for substitutional mutagenesis include thehypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in a table above as well as below.More substantial changes, denominated in amino acid classes may beintroduced and the products screened.

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (C) the bulk of the side chain. Amino acids maybe grouped according to similarities in the properties of their sidechains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75,Worth Publishers, New York (1975)):

(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp(W), Met (M)

(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn(N), Gin (Q)

(3) acidic: Asp (D), Glu (E)

(4) basic: Lys (K), Arg (R), His (H)

Alternatively, naturally occurring residues may be divided into groupsbased on other common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions typically entail exchanging a member ofone of these classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g. 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The antibodyvariants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene Ill product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and human tau. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosarnine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody are described in US Pat Appl No US 2003/0157108 A1, Presta, L.See also U.S. 2004/0693621 A1 (Kyoga Hakko Kogyo Co., Ltd). Antibodieswith a bisecting N-acetylglucosamine (GlcNAc) in the carbohydrateattached to an Fc region of the antibody are referenced in WO03/011878,Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al.

Antibodies with at least one galactose residue in the oligosaccharideattached to an Fc region of the antibody are reported in WO97/30087,Patel et al. See, also, WO98/58964 (Raju, S.) and WO99/22764 (Raju, S.)concerning antibodies with altered carbohydrate attached to the Fcregion thereof.

It may be desirable to modify the half-life of the antibodies ortau-binding fragments of the disclosures. In one embodiment, one or moreFc amino acids are mutated to increase the half-life of the antibody inthe blood, or wherein one or more sugar moieties of the Fc have beendeleted, or one or more sugar moieties added to increase the bloodhalf-life of the antibody. Common plasma proteins such as human serumalbumin (HSA) and immunoglobulins (Igs), including humanized antibodies,show long half-lifes, typically of 2 to 3 weeks, which is attributableto their specific interaction with the neonatal Fc receptor (FcRn),which leads to endosomal recycling (Ghetie (2002) Immunol Res,25:97-113). In contrast, most other proteins of pharmaceutical interest,in particular recombinant antibody fragments, hormones, and interferonssuffer from rapid (blood) clearance. This is particularly true forproteins whose size is below the threshold value for kidney filtrationof about 70 kDa (Caliceti (2003) Adv Drug Deliv Rev 55:1261-1277). Inthese cases the plasma half-life of an unmodified pharmaceutical proteinmay be considerably less than an hour. This can limit their use in mosttherapeutic applications. In order to achieve sustained pharmacologicalaction and also improved patient compliance—with required dosingintervals extending to several days or even weeks—several strategieshave been established and described in the art for purposes ofbiopharmaceutical drug development.

In other embodiments, the antibodies or tau-binding fragments thereofmay be modified to affect half-life or circulation time throughPEGylation or other conjugation to other polymers. The polymer may be ofany molecular weight, and may be branched or unbranched. Forpolyethylene glycol, the preferred molecular weight is between about 1kDa and about 100 kDa (the term “about” indicating that in preparationsof polyethylene glycol, some molecules will weigh more, some less, thanthe stated molecular weight) for ease in handling and manufacturing.Other sizes may be used, depending on the desired therapeutic profile(e.g., the duration of sustained release desired, the effects, if any onbiological activity, the ease in handling, the degree or lack ofantigenicity and other known effects of the polyethylene glycol to atherapeutic protein or analog). For example, the polyethylene glycol mayhave an average molecular weight of about 200, 500, 1000, 1500, 2000,2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000,8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500,13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000,17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000,40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000,90,000, 95,000, or 100,000 Da, Branched polyethylene glycols aredescribed, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al.,Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., NucleosidesNucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem.10:638-646 (1999). Polyethylene glycol may be attached to proteins vialinkage to any of a number of amino acid residues. For example,polyethylene glycol can be linked to polypeptides via covalent bonds tolysine, histidine, aspartic acid, glutamic acid, or cysteine residues.One or more reaction chemistries may be employed to attach polyethyleneglycol to specific amino acid residues (e.g., lysine, histidine,aspartic acid, glutamic acid, or cysteine) or to more than one type ofamino acid residue (e.g., lysine, histidine, aspartic acid, glutamicacid, cysteine and combinations thereof).

Alternatively, antibodies or fragments thereof may have increased invivo half lives via fusion with albumin (including but not limited torecombinant human serum albumin or fragments or variants thereof (see,e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622,and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporatedby reference in their entirety)) or other circulating blood proteinssuch as transferrin or ferritin. In one embodiment, polypeptides and/orantibodies of the present invention (including fragments or variantsthereof) are fused with the mature form of human serum albumin (i.e.,amino acids 1-585 of human serum albumin as shown in FIGS. 1 and 2 of EPPatent 0 322 094) which is herein incorporated by reference in itsentirety. Polynucleotides encoding fusion proteins of the disclosure arealso encompassed by the invention.

Antibodies or tau-binding fragments thereof may also be chemicallymodified to provide additional advantages such as increased solubility,stability and circulating time (in vivo half-life) of the polypeptide,or decreased immunogenicity (See, e.g. U.S. Pat. No. 4,179,337). Thechemical moieties for derivatization may be selected from water solublepolymers such as polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and thelike. The antibodies and tau-binding fragments thereof may be modifiedat random positions within the molecule, or at predetermined positionswithin the molecule and may include one, two, three or more attachedchemical moieties

It may be desirable to modify the antibodies or tau-binding fragments ofthe disclosures with respect to effector function, e.g, so as to enhanceantigen-dependent cell-mediated cyotoxicity (ADCC) and/or complementdependent cytotoxicity (CDC) of the antibody. This may be achieved byintroducing one or more amino acid substitutions in an Fc region of theantibody. Alternatively or additionally, cysteine residue(s) may beintroduced in the Fc region, thereby allowing interchain disulfide bondformation in this region. The homodimeric antibody thus generated mayhave improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al, J. Exp Med. 176:1191-1195 (1992)and Shapes, B. J. Immunol. 148:2918-2922 (1992). Alternatively, anantibody can be engineered which has dual Fc regions and may therebyhave enhanced complement lysis and ADCC capabilities. See Stevenson etal. Anti-Cancer Drug Design 3:219-230 (1989).

WO00/42072 (Presta, L.) describes antibodies with improved ADCC functionin the presence of human effector cells, where the antibodies compriseamino acid substitutions in the Fc region thereof. Preferably, theantibody with improved ADCC comprises substitutions at positions 298,333, and/or 334 of the Fc region. Preferably the altered Fc region is ahuman IgG1 Fc region comprising or consisting of substitutions at one,two or three of these positions.

Antibodies with altered C1q binding and/or complement dependentcytotoxicity (CDC) are described in WO99/51642, U.S. Pat. No.6,194,551B1, U.S. Pat. No. 6,242,195B1, U.S. Pat. No. 6,528,624B1 andU.S. Pat. No. 6,538,124 (Idusogie et al.). The antibodies comprise anamino acid substitution at one or more of amino acid positions 270, 322,326, 327, 329, 313, 333 and/or 334 of the Fc region thereof.

To increase the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, orIgG4) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

Antibodies with improved binding to the neonatal Fc receptor (FcRn), andincreased half-lives, are described in WO00/42072 (Presta, L.). Theseantibodies comprise a Fc region with one or more substitutions thereinwhich improve binding of the Fc region to FcRn. For example, the Fcregion may have substitutions at one or more of positions 238, 256, 265,272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378,380, 382, 413, 424 or 434. The preferred Fc region-comprising antibodyvariant with improved FcRn binding comprises amino acid substitutions atone, two or three of positions 307, 380 and 434 of the Fc regionthereof.

In one embodiment, the humanized antibody or a tau-bindingfragment/portion thereof is monoclonal. The monoclonal antibodies (mAbs)described herein are antibodies obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Monoclonalantibodies are highly specific, being directed against a singleantigenic site. Each mAb is directed against a single determinant(epitope) on the antigen. In addition to their specificity, themonoclonal antibodies are advantageous in that they can be synthesizedby hybridoma culture, uncontaminated by other immunoglobulins. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made in an immortalized Bcell or hybridoma thereof, or may be made by recombinant DNA methods. Inother cases, the monoclonal antibodies are made from the growth of asingle cell clone, such as a eukaryotic host cell transfected with a DNAmolecule encoding for the antibody or a tau-binding fragment or portionthereof. Nucleic acids encoding antibodies and fragments of thedisclosure can also be delivered to a host subject for expression of theantibody and fragments by cells of the host subject. Examples ofstrategies for polynucleotide delivery to and expression of anti-senilinantibodies in the central nervous system of a host subject are describedin PCT application No. WO98/44955, published Oct. 15, 1998. Any nucleicacid may be modified to increase stability in vivo. Possiblemodifications include, but are not limited to, the addition of flankingsequences at the 5′ and/or 3′ ends; the use of phosphorothioate or 2′O-methyl rather than phosphodiesterase linkages in the backbone; and/orthe inclusion of nontraditional bases such as inosine, queuosine andwybutosine, as well as acetyl-, methyl-, thio- and other modified formsof adenine, cytidine, guanine, thymine and uridine.

In one aspect, an antibody or tau-binding fragment as described hereinis capable of displaying a higher affinity for pathological tau than forphysiological tau.

In another aspect, an antibody or tau-binding fragment as describedherein is capable of inhibiting tau-tau aggregation.

In another aspect, an antibody or tau-binding fragment as describedherein is capable of mediating uptake and degradation of pathologicaltau protein by microglia.

In one aspect, an antibody or tau-binding fragment as described hereinis capable of displaying a higher affinity for pathological tau than forphysiological tau and inhibiting tau-tau aggregation.

In one aspect, an antibody or tau-binding fragment as described hereinis capable of displaying a higher affinity for pathological tau than forphysiological tau, inhibiting tau-tau aggregation, and mediating uptakeand degradation of pathological tau protein by microglia.

The tables 7, 8, and 9 below summarize the nucleic acid sequences of theCDRs and variable regions and complete chains of some of the antibodiesdescribed herein:

TABLE 7 VARIABLE NUCLEIC ACID SEQ ID NO. REGION SEQ ID NO. CDR-H1 84HEAVY DC8E8 90 CDR-H2 85 LIGHT DC8E8 91 CDR-H3 86 HEAVY 92 CHIMERICCDR-L1 87 LIGHT 93 CHIMERIC CDR-L2 88 HEAVY DC8E8 94 (identical to 92)OPTIMIZED CDR-L3 89 LIGHT DCBE8 95 (identical to 93) OPTIMIZED

TABLE 8 HUMANIZED HUMANIZED VARIABLE HEAVY VARIABLE LIGHT REGION ONLYSEQ ID NO. REGION ONLY SEQ ID NO. RHA 96 RKA 109 RHB 97 RKB 110 RHC 98RHD 99 RHE 100 RHF 101 RHG 102 RHH 103 RHI 104 RHJ 105 RHK 106 RHL 107RHM 108

TABLE 9 COMPLETE COMPLETE VARIABLE VARIABLE HEAVY CON- HEAVY CON- STANTIgG1 SEQ ID NO. STANT IgG4 SEQ ID NO. RHA 111 RHA 127 RHB 112 RHB 128RHC 113 RHC 129 RHD 114 RHD 130 RHE 115 RHE 131 RHF 116 RHF 132 RHG 117RHG 133 RHH 118 RHH 134 RHI 119 RHI 135 RHJ 120 RHJ 136 RHK 121 RHK 137RHL 122 RHL 138 RHM 123 RHM 139 cDC8E8 124 cDC8E8 140 Mouse DC8E8 125Codon optimized 126 (identical mouse DC8E8 to 124)

In a different set of embodiments, the invention provides nucleic acidsencoding the antibodies described herein, or parts thereof. The nucleicacids encoding heavy chain variable regions DC8E8 VH, RHA, RHB, RHC,RHD, RHE, RHF, RHG, RHH, RHI, RHJ, RHK, RHL, and RHM and light chainvariable regions DC8E8 VK, RKA, and RKB have been codon optimized, usingGeneScript's proprietary technology. In the context of this application,codon optimization is the process of modifying a nucleotide sequence ina manner that improves its expression, G/C content, RNA secondarystructure, and translation in eukaryotic cells, without altering theamino acid sequence it encodes.

In one embodiment, the invention provides nucleic acids (DNA or RNA)that encode a humanized antibody heavy chain variable region asdescribed herein. In one embodiment, the nucleic acid comprises a DNAthat encodes any one of heavy chain variable regions RHA, RHB, RHC, RHD,RHE, RHF, RHG, RHH, RHI, RHJ, RHK, RHL, and RHM. These nucleic acids arerepresented in the tables below. Nucleic acids exhibiting a percentidentity of at least 80%, at least 85%, at least 90%, at least 95%, andat least 98% are also within the scope of this embodiment.

The table 10 below summarizes the various nucleic acid sequencescorresponding to the various full length sequences of the light chainsof the humanized antibodies described herein.

TABLE 10 COMPLETE HUMANIZED VARIABLE LIGHT CONSTANT K SEQ ID NO. RKA 141RKB 142 cDC8E8 kappa 143

In another embodiment, the invention provides nucleic acids (DNA or RNA)that encode a humanized antibody light chain variable region asdescribed herein. In one embodiment, the nucleic acid comprises a DNAthat encodes any one of light chain variable regions RKA and RKB. Thesenucleic acids are represented in the tables above. Nucleic acidsexhibiting a percent identity of at least 80%, at least 85%, at least90%, at least 95%, and at least 98% are also within the scope of thisembodiment.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode an antibody or tau-binding fragment thereof asdescribed herein. Some of these nucleic acids bear minimal homology tothe nucleotide sequence of any “wild-type” antibody or tau-bindingfragment thereof as described herein. Nonetheless, nucleic acids thatvary due to differences in codon usage are specifically contemplated bythe present invention.

In one embodiment, the invention provides nucleic acids (DNA or RNA)that encode a humanized antibody heavy chain variable region asdescribed herein. In one embodiment, the nucleic acid comprises a DNAthat encodes any one of heavy chains variable regions RHA, RHB, RHC,RHD, RHE, RHF, RHG, RHH, RHI, RHJ, RHK, RHL, and RHM linked to a humanIgG1 constant region. Nucleic acids exhibiting a percent identity of atleast 80%, at least 85%, at least 90%, at least 95%, and at least 98%are also within the scope of this embodiment.

In one embodiment, the invention provides nucleic acids (DNA or RNA)that encode a humanized antibody heavy chain variable region asdescribed herein. In one embodiment, the nucleic acid comprises a DNAthat encodes any one of heavy chains variable regions RHA, RHB, RHC,RHD, RHE, RHF, RHG, RHH, RHI, RHJ, RHK, RHL, and RHM linked to a humanIgG4 constant region. Nucleic acids exhibiting a percent identity of atleast 80%, at least 85%, at least 90%, at least 95%, and at least 98%are also within the scope of this embodiment.

In another embodiment, the invention provides nucleic acids (DNA or RNA)that encode a humanized antibody light chain variable region asdescribed herein. In one embodiment, the nucleic acid comprises a DNAthat encodes any one of light chain variable regions RKA and RKB linkedto a human kappa region. Nucleic acids exhibiting a percent identity ofat least 80%, at least 85%, at least 90%, at least 95%, and at least 98%are also within the scope of this embodiment.

In another embodiment, the invention provides nucleic acids (DNA or RNA)that encode each of DC8E8's CDRs in a codon optimized manner.

Nucleic sequences exhibiting a percentage identity of at least 80%, forexample 85%, 90%, 95% and 98%, after optimal alignment with a preferredsequence, means nucleic sequences exhibiting, with respect to thereference nucleic sequence, certain modifications such as, inparticular, a deletion, a truncation, an extension, a chimeric fusionand/or a substitution, notably punctual. In some embodiments, these aresequences which code for the same amino acid sequences as the referencesequence, this being related to the degeneration of the genetic code, orcomplementarity sequences that are likely to hybridize specifically withthe reference sequences, for example under highly stringent conditions,notably those defined below.

Hybridization under highly stringent conditions means that conditionsrelated to temperature and ionic strength are selected in such a waythat they allow hybridization to be maintained between twocomplementarity DNA fragments. On a purely illustrative basis, thehighly stringent conditions of the hybridization step for the purpose ofdefining the polynucleotide fragments described above are advantageouslyas follows.

DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1)prehybridization at 42° C. for three hours in phosphate buffer (20 mM,pH 7.5) containing 5×SSC (1×SSC corresponds to a solution of 0.15 MNaCl+0.015 M sodium citrate), 50% formamide, 7% sodium dodecyl sulfate(SDS), 10×Denhardt's, 5% dextran sulfate and 1% salmon sperm DNA; (2)primary hybridization for 20 hours at a temperature depending on thelength of the probe (i.e.: 42° C. for a probe>100 nucleotides in length)followed by two 20-minute washings at 20° C. in 2×SSC+2% SDS, one20-minute washing at 20° C. in 0.1×SSC+0.1% SDS. The last washing iscarried out in 0.1×SSC+0.1% SDS for 30 minutes at 60° C. for a probe>100nucleotides in length. The highly stringent hybridization conditionsdescribed above for a polynucleotide of defined size can be adapted by aperson skilled in the art for longer or shorter oligonucleotides,according to the procedures described in Sambrook, et al. (Molecularcloning: a laboratory manual, Cold Spring Harbor Laboratory; 3rdedition, 2001).

Expression System

The antibodies and tau-binding fragments thereof can be madesynthetically or by any of the multiple expression systems known tothose of ordinary skill in the art. To this end, also provided areembodiments that cover cloning vectors, expression vectors, host cells,and transgenic animals (other than human) transfected, transformed, orotherwise genetically or recombinantly modified to contain one or moreof the nucleic acid sequences described above. In one embodiment, thehost cells are eukaryotic. In another embodiment, the host cells areprokaryotic. In another embodiment, the host cells express one or moreof the antibodies and tau binding fragments thereof. Selected cells maybe cultured and if required, the protein product of the gene of interestisolated from the culture using conventional techniques. In someembodiments, the expression systems have been adapted to express theantibody or tau-binding fragment thereof at an optimal level. In someembodiments, the antibodies and tau-binding fragments thereof areexpressed by contract-based antibody expression companies (e.g., Lonza)that use proprietary technologies to express the antibodies andtau-binding fragments thereof as a service. In another embodiment, theantibodies and tau-binding fragments thereof are expressed in acell-free system.

Examples of routinely used antibody expression systems includerecombinant baculovirus, lentivirus, protozoa (e.g., eukaryotic parasiteLeishmania tarentolae), microbial expression systems, includingyeast-based (e.g. Pichia pastoris, Saccharomyces cerevisiae, lipolytica,Hansenula polymorpha, Aspergillus and Trichoderma Fungi) andbacterial-based (e.g. E. coli, Pseudomonas fluorescens, Lactobacillus,Lactococcus, Bacillus megaterium, Bacillus subtilis, Brevibacillus,Corynebacterium glutamicum), Chinese hamster ovary (CHO) cells,CHOK1SVNSO (Lonza), BHK (baby hamster kidney), PerC.6 or Per.C6 (e.g.,Percivia, Crucell), different lines of HEK 293, Expi293F™ cells (LifeTechnologies), GenScript's YeastHIGH™ Technology (GenScript), humanneuronal precursor cell line AGE1.HN (Probiogen) and other mammaliancells, plants (e.g., corn, alfalfa, and tobacco), insect cells, avianeggs, algae, and transgenic animals (e.g., mice, goats, sheep, pigs,cows).

The glycosylation pattern of the antibodies expressed by these varioussystems varies considerably with the expression system. For example, theglycosylation machinery of CHO cells is somewhat similar to the humanglycosylation machinery, with some differences. In addition to thechoice of host cells, factors that affect glycosylation duringrecombinant production of antibodies include growth mode, mediaformulation, culture density, oxygenation, pH, purification schemes andthe like. Various methods have been proposed to alter the glycosylationpattern achieved in a particular host organism including introducing oroverexpressing certain enzymes involved in oligosaccharide production(U.S. Pat. Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation, orcertain types of glycosylation, can be enzymatically removed from theglycoprotein, for example using endoglycosidase H (Endo H). In addition,the recombinant host cell can be genetically engineered to be defectivein processing certain types of polysaccharides. These and similartechniques are well known in the art and can be used to alter theantibodies and tau-binding fragments thereof described herein.

The advantages and disadvantages of these various systems have beenreviewed in the literature and are known to one of ordinary skill in theart. Some of these have been described in the following references, allof which are incorporated herein by reference in their entirety: Chaddet al. Therapeutic antibody expression technology. Curr Opin Biotechnol.2001 April; 12(2):188-94; Ma et al. Human antibody expression intransgenic rats: comparison of chimeric IgH loci with human VH, D and JHbut bearing different rat C-gene regions, J Immunol Methods. 2013 Dec.31; 400-401:78-86; Zhang et al, Monoclonal antibody expression inmammalian cells. Methods Mol Biol. 2012; 907:341-58.

Pharmaceutical Compositions and Formulations

Provided herein are compositions comprising a humanized antibody or atau-binding fragment thereof, as described herein, and anothercomponent, such as a carrier. Also provided are pharmaceuticalcompositions/therapeutic formulations comprising a humanized antibody ora tau-binding fragment thereof, as described herein, and an excipientand/or a pharmaceutical carrier. In one embodiment, the carrier is not anaturally existing compound. In one embodiment, the excipient is not anaturally existing compound. In another embodiment, the diluent is not anaturally existing compound. In another embodiment, the formulationcomprising the humanized antibody or a tau-binding fragment thereof, asdescribed herein, does not contain a naturally existing compound,except, optionally, water.

In one embodiment, therapeutic formulations of the antibodies used inaccordance with the present invention are prepared for storage and/oradministration by mixing an antibody our tau-binding fragment thereofhaving the desired degree of purity with optional pharmaceuticallyacceptable carriers, diluents, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. In one embodiment,acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN, PLURONICS or polyethylene glycol (PEG).Examples of lyophilized antibody formulations are described in WO97/04801, expressly incorporated herein by reference.

In a further embodiment, the formulation further comprises a surfactant.The surfactant may, for example, be selected from a detergent,ethoxylated castor oil, polyglycolyzed glycerides, acetylatedmonoglycerides, sorbitan fatty acid esters,polyoxypropylene-polyoxyethylene block polymers (eg. poloxamers such asPluronic® F68, poloxamer 188 and 407, Triton X-100), polyoxyethylenesorbitan fatty acid esters, polyoxyethylene and polyethylene derivativessuch as alkylated and alkoxylated derivatives (tweens, e.g. Tween-20,Tween-40, Tween-80 and Brij-35), monoglycerides or ethoxylatedderivatives thereof, diglycerides or polyoxyethylene derivativesthereof, alcohols, glycerol, lectins and phospholipids (eg. phosphatidylserine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidylinositol, diphosphatidyl glycerol and sphingomyelin), derivates ofphospholipids (eg. dipalmitoyl phosphatidic acid) and lysophospholipids(eg. palmitoyl lysophosphatidyl-L-serine and1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine orthreonine) and alkyl, alkoxyl (alkyl ester), alkoxy (alkylether)-derivatives of lysophosphatidyl and phosphatidylcholines, e.g.lauroyl and myristoyl derivatives of lysophosphatidylcholine,dipalmitoylphosphatidylcholine, and modifications of the polar headgroup, that is cholines, ethanolamines, phosphatidic acid, serines,threonines, glycerol, inositol, and the positively charged DODAC, DOTMA,DCP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine, andglycerophospholipids (eg. cephalins), glyceroglycolipids (eg.galactopyransoide), sphingoglycolipids (eg. ceramides, gangliosides),dodecylphosphocholine, hen egg lysolecithin, fusidic acidderivatives—(e.g. sodium tauro-dihydrofusidate etc.), long-chain fattyacids and salts thereof C6-C12 (eg. oleic acid and caprylic acid),acylcarnitines and derivatives, N alpha.-acylated derivatives of lysine,arginine or histidine, or side-chain acylated derivatives of lysine orarginine, N alpha.-acylated derivatives of dipeptides comprising anycombination of lysine, arginine or histidine and a neutral or acidicamino acid, Nalpha-acylated derivative of a tripeptide comprising anycombination of a neutral amino acid and two charged amino acids, DSS(docusate sodium, CAS registry no [577-11-7]), docusate calcium, CASregistry no [128-49-4]), docusate potassium, CAS registry no[7491-09-0]), SDS (sodium dodecyl sulphate or sodium lauryl sulphate),sodium caprylate, cholic acid or derivatives thereof, bile acids andsalts thereof and glycine or taurine conjugates, ursodeoxycholic acid,sodium cholate, sodium deoxycholate, sodium taurocholate, sodiumglycocholate, N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,anionic (alkyl-aryl-sulphonates) monovalent surfactants, zwitterionicsurfactants (e.g. N-alkyl-N,N-dimethylammonio-1-propanesulfonates,3-cholamido-1-propyldimethylammonio-1-propanesulfonate, cationicsurfactants (quaternary ammonium bases) (e.g. cetyl-trimethylammoniumbromide, cetylpyridinium chloride), non-ionic surfactants (eg. Dodecyl.beta.-D-glucopyranoside), poloxamines (eg. Tetronic's), which aretetrafunctional block copolymers derived from sequential addition ofpropylene oxide and ethylene oxide to ethylenediamine, or the surfactantmay be selected from the group of imidazoline derivatives, or mixturesthereof. In one embodiment, the surfactant is not a naturally existingcompound. Each one of these specific surfactants constitutes analternative embodiment of the disclosure.

One embodiment provides for stable formulations of the antibodies and/ortau-binding fragments thereof, which comprise preferably a phosphatebuffer with saline or a chosen salt, as well as preserved solutions andformulations containing a preservative, as well as multi-use preservedformulations suitable for pharmaceutical or veterinary use, comprisingat least one the antibodies and/or tau-binding fragments thereof in apharmaceutically acceptable formulation. In one embodiment, preservedformulations contain at least one known preservative or optionallyselected from the group consisting of at least one phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuricnitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride(e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and thelike), benzalkonium chloride, benzethonium chloride, sodiumdehydroacetate and thimerosal, or mixtures thereof in an aqueousdiluent. Any suitable concentration or mixture can be used as known inthe art, such as 0.001-5%, or any range or value therein, such as, butnot limited to 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1,6, 1.7, 1,8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5,4.6, 4.7, 4.8, 4.9, or any range or value therein. Non-limiting examplesinclude, no preservative, 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5,0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0,2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol(e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s)(e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02,0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.In one embodiment, the preservative or preservatives are not naturallyexisting compounds.

In one embodiment, the antibodies and tau-binding fragments thereof ofthe disclosure can be incorporated into pharmaceutical compositionssuitable for administration to a subject. In one common embodiment, thepharmaceutical composition comprises an antibody or tau-binding fragmentthereof of the invention and a pharmaceutically acceptable carrier. Inone embodiment, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Additional examples of pharmaceuticallyacceptable carriers include one or more of water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the antibodyor tau-binding fragment thereof.

In one embodiment, an appropriate amount of apharmaceutically-acceptable salt is used in the formulation to renderthe formulation isotonic. Examples of the carrier include saline,Ringer's solution and dextrose solution. In one embodiment, the pH ofthe solution is from about 5 to about 8. In another embodiment, the pHis from about 7 to about 7.5. Further carriers include sustained releasepreparations such as semipermeable matrices of solid hydrophobicpolymers containing the antibody or tau-binding fragment thereof, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. A sustained release matrix, as used herein, is a matrixmade of materials, usually polymers which are degradable by enzymatic oracid/base hydrolysis or by dissolution. Once inserted into the body, thematrix is acted upon by enzymes and body fluids. The sustained releasematrix desirably is chosen by biocompatible materials such as liposomes,polylactides (polylactide acid), polyglycolide (polymer of glycolicacid), polylactide co-glycolide (copolymers of lactic acid and glycolicacid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid,collagen, chondroitin sulfate, carboxylic acids, fatty acids,phospholipids, polysaccharides, nucleic acids, polyamino acids, aminoacids such phenylalanine, tyrosine, isoleucine, polynucleotides,polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferredbiodegradable matrix is a matrix of one of either polylactide,polyglycolide, or polylactide co-glycolide (co-polymers of lactic acidand glycolic acid).

It will be apparent to those persons skilled in the art that certaincarriers may be more preferable depending upon, for instance, the routeof administration and concentration of antibody being administered.

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories. Insome embodiments, such compositions may also comprise buffers (e.g.,neutral buffered saline or phosphate buffered saline), carbohydrates(e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins,polypeptides or amino acids such as glycine, antioxidants, chelatingagents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide)and/or preservatives. Alternatively, compositions of the presentinvention may be formulated as a lyophilizate. Antibodies andtau-binding fragments thereof may also be encapsulated within liposomesusing well-known technologies.

Dosage forms suitable for internal administration generally contain fromabout 0.1 milligram to about 500 milligrams of antibody or tau-bindingfragment thereof (the active ingredient) per unit or container. In thesepharmaceutical compositions, the active ingredient will ordinarily bepresent in an amount of about 0.5-99.999% by weight based on the totalweight of the composition.

Therapeutic compositions/formulations typically must be sterile andstable under the conditions of manufacture and storage. The compositioncan be formulated as a solution, microemulsion, dispersion, liposome, orother ordered structure suitable to high drug concentration. Sterileinjectable solutions can be prepared by incorporating the activecompound (i.e., antibody or tau-binding fragment thereof) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

The preferred dosage form depends on the intended mode of administrationand therapeutic application. One of ordinary skill in the art isfamiliar with the procedures for determining such dosages. Typicalcompositions are in the form of injectable or infusible solutions, suchas compositions similar to those used for passive immunization of humanswith other antibodies. The most typical mode of administration isparenteral (e.g., intravenous, subcutaneous, intraperitoneal,intramuscular). In a preferred embodiment, the antibody is administeredby intravenous infusion or injection. In another preferred embodiment,the antibody is administered by intramuscular or subcutaneous injection.

The antibodies and tau-binding fragments thereof of the presentinvention can be administered by a variety of methods known in the art,although for many therapeutic applications, the preferred route/mode ofadministration is intravenous injection or infusion. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. In certainembodiments, the antibody or tau-binding fragment thereof may beprepared with a carrier that will protect the compound against rapidrelease, such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker. Inc., New York, 1978.

In certain embodiments, an antibody or tau-binding fragment thereof ofthe invention may be orally administered, for example, with an inertdiluent or an assimilable edible carrier. The antibody or tau-bindingfragment thereof (and other ingredients, if desired) may also beenclosed in a hard or soft shell gelatin capsule, compressed intotablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the antibody or tau-binding fragment thereofmay be incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. To administer an antibody or tau-bindingfragment thereof of the invention by other than parenteraladministration, it may be necessary to coat the antibody or tau-bindingfragment thereof with, or co-administer the antibody or tau-bindingfragment thereof with, a material to prevent its inactivation.

Certain embodiments of the invention provide for the antibody ortau-binding fragment thereof to traverse the blood-brain barrier.Certain neurodegenerative diseases, including AD and relatedtauopathies, are associated with an increase in permeability of theblood-brain barrier, such that the antibody or tau-binding fragmentthereof can be readily introduced to the brain. When the blood-brainbarrier remains intact, several art-known approaches exist fortransporting molecules across it, including, but not limited to,physical methods, lipid-based methods, and receptor and channel-basedmethods.

Physical methods of transporting the antibody or tau-binding fragmentthereof across the blood-brain barrier include, but are not limited to,circumventing the blood-brain barrier entirely, or by creating openingsin the blood-brain barrier. Circumvention methods include, but are notlimited to, direct injection into the brain (see, e.g., Papanastassiouet al., Gene Therapy 9: 398-406 (2002)) and implanting a delivery devicein the brain (see, e.g., Gill et al., Nature Med. 9: 589-595 (2003); andGliadel Wafers™, Guildford Pharmaceutical). Methods of creating openingsin the barrier include, but are not limited to, ultrasound (see, e.g.,U.S. Patent Publication No. 2002/0038086), osmotic pressure (e.g., byadministration of hypertonic mannitol (Neuwelt, E. A., Implication ofthe Blood-Brain Barrier and its Manipulation, Vols 1 & 2, Plenum Press,N.Y. (1989))), permeabilization by, e.g., bradykinin or permeabilizerA-7 (see, e.g., U.S. Pat. Nos. 5,112,596, 5,268,164, 5,506,206, and5,686,416), and transfection of neurons that straddle the blood-brainbarrier with vectors containing genes encoding the antibody ortau-binding fragment thereof (see, e.g., U.S. Patent Publication No,2003/0083299).

Lipid-based methods of transporting the antibody or tau-binding fragmentthereof across the blood-brain barrier include, but are not limited to,encapsulating the antibody or tau-binding fragment thereof in liposomesthat are coupled to active fragments thereof that bind to receptors onthe vascular endothelium of the blood-brain barrier (see, e.g., U.S.Patent Application Publication No. 20020025313), and coating theantibody or tau-binding fragment thereof in low-density lipoproteinparticles (see, e.g., U.S. Patent Application Publication No.20040204354) or apolipoprotein E (see, e.g., U.S. Patent ApplicationPublication No. 20040131692).

Receptor and channel-based methods of transporting the antibody ortau-binding fragment thereof across the blood-brain barrier include, butare not limited to, using glucocorticoid blockers to increasepermeability of the blood-brain barrier (see, e.g., U.S. PatentApplication Publication Nos. 2002/0065259, 2003/0162695, and2005/0124533); activating potassium channels (see, e.g., U.S. PatentApplication Publication No. 2005/0089473), inhibiting ABC drugtransporters (see, e.g., U.S. Patent Application Publication No.2003/0073713); coating antibodies with a transferrin and modulatingactivity of the one or more transferrin receptors (see, e.g., U.S.Patent Application Publication No. 2003/0129186), and cationizing theantibodies (see, e.g., U.S. Pat. No. 5,004,697).

A variety of other approaches are known in the art to effectadministration of compounds to the brain. For example, the antibody ortau-binding fragment thereof may be administered by directintraventricular or intrathecal injection, preferably via slow infusionto minimize impact on brain parenchyma. The desired antibody ortau-binding fragment thereof may also be delivered using a slow releaseimplant in the brain, or implanted recombinant cells that produce theantibody or tau-binding fragment thereof. The blood brain barrier (BBB)may be permeabilized concomitant with the antibody or tau-bindingfragment thereof administration, to permit movement of the antibody ortau-binding fragment thereof across the BBB. Permeabilizing agentsinclude osmotic agents, such as hypertonic mannitol, or anotherpermeabilizing agent such as bradykinin, an alkylglycerol, ultrasound,electromagnetic radiation or parasympathetic innervation.

In addition, and without being bound by any specific mechanism, it hasalso been considered that it is possible that an antibody, in the blood,could have a “sink-like” effect in removing its target protein from thebrain. See, e.g., US Published Application US 20110158986, paragraph[0017]. If that is the case, the antibodies and tau-binding fragmentsthereof could be useful to remove pathological tau from the brain outinto the circulation, effectively preventing it from causing furtherdamage to the neuronal cells and tissues.

Supplementary or combination active compounds or therapeutic agents asdisclosed elsewhere in this application can also be incorporated intothe compositions, administered concurrently, or administeredsequentially with the antibody or tau-binding fragment thereof. Incertain embodiments, an antibody or tau-binding fragment thereof of theinvention is coformulated with and/or coadministered with one or moreadditional therapeutic agents. These additional therapeutic agents canalso be chemically conjugates to the antibodies or tau-binding fragmentsthereof described herein.

In one embodiment, an immunoconjugate is provided having the formula(A)-(L)-(C), wherein: (A) is an antibody or binding fragment thereof ofany one of claims 1-51; (L) is a linker; and (C) is an agent; andwherein said linker (L) links (A) to (C). In one embodiment, (C) is aneffector molecule, e.g., therapeutic agent, an imaging agent, adetectable agent, or a diagnostic agent. In some embodiments, theseconjugates are referred to herein as antibody-drug-conjugates (ADCs).

A (L) linker, as used herein, is a molecule that is used to join the (A)to (C). The linker is capable of forming covalent bonds to both theantibody and to the effector molecule. Suitable linkers are well knownto those of skill in the art and include, but are not limited to,straight or branched-chain carbon linkers, heterocyclic carbon linkers,or peptide linkers. Where the antibody and the effector molecule arepolypeptides, the linkers may be joined to the constituent amino acidsthrough their side groups (e.g., through a disulfide linkage tocysteine). However, in a preferred embodiment, the linkers will bejoined to the alpha carbon amino and carboxyl groups of the terminalamino acids.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will compriselinkages which are cleavable in the vicinity of the target site.Cleavage of the linker to release the effector molecule from theantibody may be prompted by enzymatic activity or conditions to whichthe immunoconjugate is subjected either inside the target cell or in thevicinity of the target site. In yet other embodiments, the linker unitis not cleavable and the drug is released, for example, by antibodydegradation.

A number of different reactions are available for covalent attachment ofdrugs and/or linkers to antibodies or fragments thereof. This is oftenaccomplished by reaction of the amino acid residues of the antibodymolecule, including the amine groups of lysine, the free carboxylic acidgroups of glutamic and aspartic acid, the sulfhydryl groups of cysteineand the various moieties of the aromatic amino acids. One of the mostcommonly used non-specific methods of covalent attachment is thecarbodiimide reaction to link a carboxy (or amino) group of a compoundto amino (or carboxy) groups of the antibody. Additionally, bifunctionalagents such as dialdehydes or imidoesters have been used to link theamino group of a compound to amino groups of an antibody molecule. Alsoavailable for attachment of drugs to antibodies is the Schiff basereaction. This method involves the periodate oxidation of a drug thatcontains glycol or hydroxy groups, thus forming an aldehyde which isthen reacted with the binding agent. Attachment occurs via formation ofa Schiff base with amino groups of the binding agent. Isothiocyanatescan also be used as coupling agents for covalently attaching drugs tobinding agents.

In some embodiments, the linker is cleavable by a cleaving agent that ispresent in the intracellular environment (e.g., within a lysosome orendosome or caveolea). The linker can be, e.g., a peptidyl linker thatis cleaved by an intracellular peptidase or protease enzyme, including,but not limited to, a lysosomal or endosomal protease. In someembodiments, the peptidyl linker is at least two amino acids long or atleast three amino acids long. Cleaving agents can include cathepsins Band D and plasmin, all of which are known to hydrolyze dipeptide drugderivatives resulting in the release of active drug inside target cells(see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123).Most typical are peptidyl linkers that are cleavable by enzymes that arepresent in 191P4D12-expressing cells. Other examples of such linkers aredescribed, e.g., in U.S. Pat. No. 6,214,345. In a specific embodiment,the peptidyl linker cleavable by an intracellular protease is a Val-Citlinker or a Phe-Lys linker (see, e.g., U.S. Pat. No. 6,214,345, whichdescribes the synthesis of doxorubicin with the Val-Cit linker). Oneadvantage of using intracellular proteolytic release of the therapeuticagent is that the agent is typically attenuated when conjugated and theserum stabilities of the conjugates are typically high.

In other embodiments, the cleavable linker is pH-sensitive, i.e.,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker hydrolyzable under acidic conditions. For example,an acid-labile linker that is hydrolyzable in the lysosome (e.g., ahydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,orthoester, acetal, ketal, or the like) can be used. (See, e.g., U.S.Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999,Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem.264:14653-14661.) Such linkers are relatively stable under neutral pHconditions, such as those in the blood, but are unstable at below pH 5.5or 5.0, the approximate pH of the lysosome. In certain embodiments, thehydrolyzable linker is a thioether linker (such as, e.g., a thioetherattached to the therapeutic agent via an acylhydrazone bond (see, e.g.,U.S. Pat. No. 5,622,929).

In yet other embodiments, the linker is cleavable under reducingconditions (e.g., a disulfide linker). A variety of disulfide linkersare known in the art, including, for example, those that can be formedusing SATA (N-succinimidyl-S-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-,SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.47:5924-5931; Wawrzynczak et al., In Immunoconjugates: AntibodyConjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed OxfordU. Press, 1987. See also U.S. Pat. No. 4,880,935.)

In yet other specific embodiments, the linker is a malonate linker(Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyllinker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a3′-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

In yet other embodiments, the linker unit is not cleavable and the drugis released by antibody degradation. (See U.S. Publication No,2005/0238649).

In one embodiment, the linker is not substantially sensitive to theextracellular environment. As used herein, “not substantially sensitiveto the extracellular environment,” in the context of a linker, meansthat no more than about 20%, typically no more than about 15%, moretypically no more than about 10%, and even more typically no more thanabout 5%, no more than about 3%, or no more than about 1% of thelinkers, in a sample of antibody-drug conjugate compound, are cleavedwhen the antibody-drug conjugate compound presents in an extracellularenvironment (e.g., in plasma). Whether a linker is not substantiallysensitive to the extracellular environment can be determined, forexample, by incubating with plasma the antibody-drug conjugate compoundfor a predetermined time period (e.g., 2, 4, 8, 16, or 24 hours) andthen quantitating the amount of free drug present in the plasma.

In other, non-mutually exclusive embodiments, the linker promotescellular internalization. In certain embodiments, the linker promotescellular internalization when conjugated to the therapeutic agent (i.e.,in the milieu of the linker-therapeutic agent moiety of theantibody-drug conjugate compound as described herein).

A variety of exemplary linkers that can be used with the presentcompositions and methods are described in WO 2004-010957, U.S.Publication No. 2006/0074008, U.S. Publication No. 20050238649, and U.S.Publication No. 2006/0024317 (each of which is incorporated by referenceherein in its entirety and for all purposes).

Some examples of antibody-drug conjugates (ADCs) currently existing inthe clinic can be found in Feng, Y. et al. Conjugates of Small MoleculeDrugs with Antibodies and Other Proteins. Biomedicines 2014, 2, 1-13;doi:10.3390/biomedicines2010001.

In view of the large number of methods that have been reported forattaching a variety of therapeutic agents, imaging agents, detectableagents, diagnostic agents, radiodiagnostic compounds, radiotherapeuticcompounds, drugs, toxins, and other agents to antibodies and fragmentsthereof, one skilled in the art will be able to determine a suitablemethod for attaching a given agent to an antibody or tau-bindingfragment thereof. In another embodiment, (A) and (C) are directly boundto each other.

In another embodiment, (L) is a spacer group or a linkage group such aspolyaldehyde, glutaraldehyde, ethylenediaminetetraacetic acid (EDTA) ordiethylenetriaminepentaacetic acid (DPTA). Other techniques for linkingan antibody or fragment to another compound include the formation ofdisulfide linkages using N-succinimidyl-3-(2-pyridyl-thio) propionate(SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC) or derivatives (if the peptide lacks a sulfhydryl group, this canbe provided by addition of a cysteine residue). These reagents create adisulfide linkage between themselves and peptide cysteine resides on oneprotein and an amide linkage through the .epsilon.-amino on a lysine, orother free amino group in other amino acids. A variety of suchdisulfide/amide-forming agents are described by Immun. Rev. 62, 185(1982). Other bifunctional coupling agents form a thioether rather thana disulfide linkage. Many of these thio-ether-forming agents arecommercially available and include reactive esters of 6-maleimidocaproicacid, 2-bromoacetic acid, and 2-iodoacetic acid,4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid. The carboxyl groupscan be activated by combining them with succinimide or1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.

Drug loading is represented by p and is the average number of Drugmoieties per antibody in a molecule (e.g., A-L-Dp). Drug loading mayrange from 1 to 20 drug moieties (D) per antibody, ADCs of the inventioninclude collections of antibodies conjugated with a range of drugmoieties, from 1 to 20. The average number of drug moieties per antibodyin preparations of ADC from conjugation reactions may be characterizedby conventional means such as mass spectroscopy and, ELISA assay. Thequantitative distribution of ADC in terms of p may also be determined.In some instances, separation, purification, and characterization ofhomogeneous ADC where p is a certain value from ADC with other drugloadings may be achieved by means such as electrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in the exemplary embodiments above, an antibody mayhave only one or several cysteine thiol groups, or may have only one orseveral sufficiently reactive thiol groups through which a linker may beattached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the drug loading for an ADC of the invention ranges from 1to about 8; from about 2 to about 6; from about 3 to about 5; from about3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8;from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3to about 3.8; or from about 3.3 to about 3.7. Indeed, it has been shownthat for certain ADCs, the optimal ratio of drug moieties per antibodymay be less than 8, and may be about 2 to about 5. See U.S. Pat. No.7,498,298.

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, e.g., by: (i) limiting the molar excess of drug-linkerintermediate or linker reagent relative to antibody, (ii) limiting theconjugation reaction time or temperature, (iii) partial or limitingreductive conditions for cysteine thiol modification, (iv) engineeringby recombinant techniques the amino acid sequence of the antibody suchthat the number and position of cysteine residues is modified forcontrol of the number and/or position of linker-drug attachments (suchas thioMab or thioFab prepared as disclosed herein and in WO2006/034488.

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of one or more drug moieties attached toan antibody. The average number of drugs per antibody may be calculatedfrom the mixture by a dual ELISA antibody assay, which is specific forantibody and specific for the drug. Individual ADC molecules may beidentified in the mixture by mass spectroscopy and separated by HPLC,e.g. hydrophobic interaction chromatography (see, e.g., Hamblett, K J.,et al. “Effect of drug loading on the pharmacology, pharmacokinetics,and toxicity of an anti-CD30 antibody-drug conjugate,” Abstract No. 624,American Association for Cancer Research, 2004 Annual Meeting, Mar.27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley, S.C., et al. “Controlling the location of drug attachment in antibody-drugconjugates,” Abstract No. 627, American Association for Cancer Research,2004 Annual Meeting, Mar. 27-31, 2004, Proceedings of the AACR, Volume45, March 2004). In certain embodiments, a homogeneous ADC with a singleloading value may be isolated from the conjugation mixture byelectrophoresis or chromatography.

In some exemplary embodiments, an antibody or tau-binding fragmentthereof described herein may be coformulated and/or coadministered withone or more additional compounds that are also useful in the prophylaxisand/or treatment of AD. These include, without limitation, compoundsthat are useful in active and passive immunotherapies for AD, such asbeta-amyloid peptides (e.g., N-terminal amyloid beta peptides), taupeptides, which might or might not be conjugated to other compounds,such as mutated diphtheria toxin; antibodies against beta-amyloid, suchas bapineuzumab, solaneuzumab, gantenerumab, crenezumab, ponezumab, andIVIG immunoglobulin, other immunization therapies targeting Abetaoligomers, other tau antibodies, compounds preventing thehyperphosphorylation of tau, and other active and passive immunizationtherapies targeting tau aggregates Other drugs that should be helpful incombination therapy with the antibodies and tau-binding fragmentsdescribed herein are amyloid-beta aggregation inhibitors (e.g.,Tramiprosate), gamma-secretase inhibitors (e.g., semagacestat), andgamma-secretase modulators (tarenflurbil). Furthermore, one or moreantibodies of the invention may be used in combination with two or moreof the foregoing therapeutic agents. At early stages of the disease,combination therapies can be advantageous. Combination therapies arealso advantageous at later stages of the disease, such as combination ofhAb and growth factors and other biologically active molecules inducingneuronal plasticity and regeneration. Such combination therapies mayadvantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible toxicities or complications associatedwith the various monotherapies. According to a related embodiment, anantibody or tau-binding fragment thereof described herein is used incombination (one administered separately, before, simultaneously with orafter the other) with at least one combination agent chosen fromacetylcholinesterase inhibitors (e.g., donepezil, rivastigmine,galantamine, tacrine, nutritive supplements), N-Methyl-D-aspartate(NMDA) receptor antagonists (e.g., memantine), inhibitors of DNA repair(e.g., pirenzepine or a metabolite thereof), transition metal chelators,growth factors, hormones, non-steroidal anti-inflammatory drugs (NSAID),antioxidants, lipid lowering agents, selective phosphodiesteraseinhibitors, inhibitors of tau aggregation, inhibitors of proteinkinases, inhibitors of anti-mitochondrial dysfunction drugs,neurotrophins, inhibitors of heat shock proteins, inhibitors ofLipoprotein-associated phospholipase A₂, and any pharmaceuticallyacceptable salts thereof. In one embodiment, the treatment with anantibody and/or tau-binding fragment thereof is combined with treatmentwith cholinesterase inhibitors (ChEl) and/or memantine, which offermodest symptomatic benefit. In one embodiment, the combinationtherapeutic agent is selected from the group consisting of ananti-apoptotic compound, a metal chelator, an inhibitor of DNA repair,3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate (1,3PDS),a secretase activator, a beta-secretase inhibitor, a gamma-secretaseinhibitor, a beta-amyloid peptide, a beta-amyloid antibody, aneurotransmitter, a beta-sheet breaker, an anti-inflammatory molecule,and a cholinesterase inhibitor. In one embodiment, the cholinesteraseinhibitor is tacrine, rivastigmine, donepezil, galantamine, or anutritive supplement. In another embodiment, the additional therapeuticagent is selected from BACE inhibitors; muscarinic antagonists;cholinesterase inhibitors; gamma secretase inhibitors; gamma secretasemodulators; HMG-CoA reductase inhibitors; non-steroidalanti-inflammatory agents; N-methyl-D-aspartate receptor antagonists;anti-amyloid antibodies; vitamin E; nicotinic acetylcholine receptoragonists; CB1 receptor inverse agonists or CB1 receptor antagonists; anantibiotic; growth hormone secretagogues; histamine H3 antagonists; AMPAagonists; PDE4 inhibitors; GABA_(A) inverse agonists; inhibitors ofamyloid aggregation; glycogen synthase kinase beta inhibitors; promotersof alpha secretase activity; PDE-10 inhibitors and cholesterolabsorption inhibitors.

Other compounds that can be suitably used in combination with theantibody and tau-binding fragment described herein are described in WO2004/058258 (see especially pages 16 and 17) including therapeutic drugtargets (page 36-39), alkanesulfonic acids and alkanolsulfuric acids(pages 39-51), cholinesterase inhibitors (pages 51-56), NMDA receptorantagonists (pages 56-58), estrogens (pages 58-59), non-steroidalanti-inflammatory drugs (pages 60-61), antioxidants (pages 61-62),peroxisome proliferators-activated receptor (PPAR) agonists (pages63-67), cholesterol-lowering agents (pages 68-75); amyloid inhibitors(pages 75-77), amyloid formation inhibitors (pages 77-78), metalchelators (pages 78-79), anti-psychotics and anti-depressants (pages80-82), nutritional supplements (pages 83-89) and compounds increasingthe availability of biologically active substances in the brain (seepages 89-93) and prodrugs (pages 93 and 94).

In one embodiment, the antibody and/or tau-binding fragment thereof isused in combination with the current standard of treatment at the timeof treatment, which includes cholinesterase inhibitors and memantine(Namenda) NMDA antagonist.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or tau-binding fragment thereof of the invention.A “therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor tau-binding fragment thereof may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the antibody or tau-binding fragment thereof to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of the antibody ortau-binding fragment thereof are outweighed by the therapeuticallybeneficial effects. A “prophylactically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired prophylactic result. Typically, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, an infusionprotocol, or a single bolus, may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationsfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic or prophylactic effect to be achieved, and(b) the limitations inherent in the art of compounding such an activecompound for the treatment of sensitivity in individuals.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

For example, effective doses of the compositions of the presentdisclosure, for the treatment of the conditions described below, varydepending upon many factors, including means of administration, targetsite, physiological state of the patient, whether the patient is humanor an animal, other medications administered, and whether the treatmentis prophylactic or therapeutic. Usually, the patient is a human.Treatment dosages need to be titrated to optimize safety and efficacy.Accordingly, treatment with an antibody or tau-binding fragment thereofwill typically entail multiple dosages over a period of time.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or tau-binding fragmentthereof of the invention for humans is 0.1-20 mg/kg, more preferably1-10 mg/kg. In one preferred embodiment, the antibody is administered inmultiple dosages over a period of at least three months, preferably atleast six months, and a dose between 1 and 10 mg/kg. Optionally theantibody or tau-binding fragment thereof is administered at a dose of0.01-0.6 mg/kg and a frequency of between weekly and monthly.Optionally, the antibody or tau-binding fragment thereof is administeredat a dose of 0.05-0.5 mg/kg. Optionally, the antibody or tau-bindingfragment thereof is administered at a dose of 0.05-0.25 mg/kg.Optionally, the antibody or tau-binding fragment thereof is administeredat a dose of 0.015-0.2 mg/kg weekly to biweekly. Optionally, theantibody or tau-binding fragment thereof is administered at a dose of0.05-0.15 mg/kg weekly to biweekly. Optionally, the antibody ortau-binding fragment thereof is administered at a dose of 0.05-0.07mg/kg weekly. Optionally, the antibody or tau-binding fragment thereofis administered at a dose of 0.06 mg/kg weekly. Optionally, the antibodyor tau-binding fragment thereof is administered at a dose of 0.1 to 0.15mg/kg biweekly. Optionally, the antibody or tau-binding fragment thereofis administered at a dose of 0.1 to 0.3 mg/kg monthly. Optionally, theantibody or tau-binding fragment thereof is administered at a dose of0.2 mg/kg monthly. Optionally, the antibody or tau-binding fragmentthereof is administered once a year. Optionally, the antibody ortau-binding fragment thereof is administered at a dose of 1-40 mg and afrequency of between weekly and monthly. Optionally, the antibody ortau-binding fragment thereof is administered at a dose of 5-25 mg.Optionally, the antibody or tau-binding fragment thereof is administeredat a dose of 2.5-15 mg. Optionally, the antibody or tau-binding fragmentthereof is administered at a dose of 1-12 mg weekly to biweekly.Optionally, the antibody or tau-binding fragment thereof is administeredat a dose of 2.5-10 mg weekly to biweekly. Optionally, the antibody ortau-binding fragment thereof is administered at a dose of 2.5-5 mgweekly. Optionally, the antibody or tau-binding fragment thereof isadministered at a dose of 4-5 mg weekly. Optionally, the antibody ortau-binding fragment thereof is administered at a dose of 7-10 mgbiweekly.

In other passive immunization embodiments with an antibody ortau-binding fragment as described herein, the dosage ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg of the host bodyweight. In some applications, the amount of antibody or tau-bindingfragment thereof can be administered at a dosage of at least 0.1 mg/kgof body weight, at a dosage of at least 0.5 mg/kg of body weight, 1mg/kg of body weight, or any combination of dosages between 0.1 and 10mg/kg of body weight. In some methods, the antibody or tau-bindingfragment thereof can be administered in multiple dosages (equal ordifferent) over a period of at least 1 month, at least 3 months, or atleast 6 months. The total number of doses over any one treatment periodcan be, for example, between 4 and 6, although other numbers can be useddepending on the factors discussed above. Treatment can be monitored byany of the methods described further below.

Suitable antibody formulations can also be determined by examiningexperiences with other already developed therapeutic monoclonalantibodies. For example, several monoclonal antibodies have been shownto be efficient in clinical situations, such as Rituxan (Rituximab),Herceptin (Trastuzumab), Xolair (Omalizumab), Bexxar (Tositumomab),Campath (Alemtuzumab), Zevalin, Oncolym, Remicade (infliximab), Lucentis(Ranibizumab), Synagis (Palivizumab), Solids (Eculizumab), Kadcyla(ado-trastuzumab emtansine), Avastin (Bevacizumab), Erbitux (cetuximab),Simponi (Golimumab), Tysabri (natalizumab), MabThera (Rituximab),Stelara (Ustekinumab), Pritumumab, and Aducanumab, and similarformulations may be used with the antibodies of this disclosure. Forexample, a monoclonal antibody can be supplied at a concentration of 10mg/mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials,formulated for IV administration in 9.0 mg/mL sodium chloride, 7.35mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and SterileWater for Injection. The ph is adjusted to 6.5. In another embodiment,the antibody is supplied in a formulation comprising about 20 mMNa-Citrate, about 150 mM NaCl, at pH of about 6.0.

Methods of Treatment and Prophylaxis

The antibodies and compositions described herein can be used for variousmethods of treatment and prophylaxis of AD and related tauopathies. Inaddition to the advantageous property of reduced immunogenicity, theseantibodies have at least 80% of the binding affinity for disease tauthan the parent mouse DC8E8 antibody. Mouse DC8E8 was extensivelycharacterized in WO2013/041962, where it was shown that it possessestherapeutic properties in an in vivo model of AD. Thus, there is areasonable basis to believe that humanized DC8E8 will also be useful inthe therapy and prophylaxis of human AD, while potentially eliciting aless detrimental immunologic response.

In one embodiment, the method comprises administering the antibodies,nucleic acids encoding them, or pharmaceutical compositions as describedabove to the subject/patient. In prophylactic applications,pharmaceutical compositions or medicaments are administered to a patientsusceptible to, or otherwise at risk of, Alzheimer's disease or anothertauopathy in an amount sufficient to eliminate or reduce the risk,lessen the severity, or delay the outset of the disease, includingbiochemical, histologic and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. In therapeutic applications, compositions ormedicaments are administered to a patient suspected of, or alreadysuffering from such a disease in amount sufficient to cure, or at leastpartially arrest, the symptoms of the disease (biochemical, histologicand/or behavioral), including its complications and intermediatepathological phenotypes in development of the disease.

As defined above, treatment encompasses the application oradministration of a therapeutic agent to a subject, who has a disease, asymptom of disease or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease, the symptoms of disease or thepredisposition toward disease. Moreover, as long as the compositions ofthe disclosure either alone or in combination with another therapeuticagent cure, heal, alleviate, relieve, alter, remedy, ameliorate, improveor affect at least one symptom of Alzheimer's Disease or anothertauopathy being treated as compared to that symptom in the absence ofuse of the humanized anti-tau antibody or tau-binding fragment thereofcomposition, the result should be considered an effective treatment ofthe underlying disorder regardless of whether all the symptoms of thedisorder are cured, healed, alleviated, relieved, altered, remedied,ameliorated, improved or affected or not

An individual “at risk” may or may not have detectable disease, and mayor may not have displayed detectable disease prior to the treatmentmethods described herein. “At risk” denotes that an individual has oneor more so-called risk factors, which are measurable parameters thatcorrelate with development of Alzheimer's disease. An individual havingone or more of these risk factors has a higher probability of developingAlzheimer's disease than an individual without these risk factor(s).These risk factors include, but are not limited to, age, sex, race,diet, history of previous disease, presence of precursor disease,genetic (i.e., hereditary) considerations, and environmental exposure,and are well-known to one of ordinary skill in the art.

In one embodiment, the disclosure provides a method of treating orpreventing the progression of Alzheimer's disease or another tauopathyin a subject, the method comprising administering to said subject aneffective amount of at least one antibody and/or tau-binding fragmentthereof as provided herein. In some embodiments, the method is capableof reducing motor impairment, improving motor function, reducingcognitive impairment, improving cognitive function, or a combinationthereof.

In other embodiments, the disclosure provides a method of amelioratingat least one of the symptoms associated with Alzheimer's disease oranother tauopathy in a subject, the method comprising administering tosaid subject an effective amount of at least one antibody and/ortau-binding fragment thereof as provided herein.

In one embodiment, the disclosure provides a method of delaying theprogression of Alzheimer's disease. In one embodiment, “delaying”development of Alzheimer's disease means to defer, hinder, slow, retard,stabilize, and/or postpone development of the disease. This delay can beof varying lengths of time, depending on the history of the diseaseand/or individual being treated. As is evident to one skilled in theart, a sufficient or significant delay can, in effect, encompassprevention, in that the individual does not develop the disease. In oneembodiment, a method that “delays” development of Alzheimer's disease isa method that reduces probability of disease development in a given timeframe and/or reduces extent of the disease in a given time frame, whencompared to not using the method. Such comparisons are typically basedon clinical studies, using a statistically significant number ofsubjects.

Patients, subjects, or individuals include mammals, such as human,bovine, equine, canine, feline, porcine, and ovine animals. The subjectis preferably a human, and may or may not be afflicted with disease orpresently show symptoms. In the case of Alzheimer's disease, virtuallyanyone is at risk of suffering from Alzheimer's disease if he or shelives long enough. Therefore, the present methods can be administeredprophylactically to the general population without the need for anyassessment of the risk of the subject patient.

In one embodiment, the patient herein is optionally subjected to adiagnostic test prior to therapy. In one embodiment, the present methodsare useful for individuals who do have a known genetic risk ofAlzheimer's disease. Such individuals include those having relatives whohave experienced this disease, and those whose risk is determined byanalysis of genetic or biochemical markers. Genetic markers of risktoward Alzheimer's disease include mutations in the APP gene,particularly mutations at position 717 and positions 670 and 671referred to as the Hardy and Swedish mutations respectively (see Hardy(1997) Trends Neurosci. 20:154-9). Other markers of risk are mutationsin the presenilin genes, PS1 and PS2, and ApoE4, family history of AD,hypercholesterolemia or atherosclerosis. Individuals presently sufferingfrom Alzheimer's disease can be recognized from characteristic dementia,as well as the presence of risk factors described above. In addition, anumber of diagnostic tests are available for identifying individuals whohave AD. These include measurement of CSF tau and Amyloid-beta42 levels.Elevated tau and decreased Amyloid-beta42 levels signify the presence ofAD. In one embodiment, individuals suffering from Alzheimer's diseasecan be diagnosed by ADRDA (Alzheimer's Disease and Related DisordersAssociation) criteria. In asymptomatic patients, treatment can begin atany age (e.g., 10, 20, 30). Usually, however, it is not necessary tobegin treatment until a patient reaches 40, 50, 60 or 70. Treatmenttypically entails multiple dosages over a period of time. Treatment canbe monitored by various ways known in the art over time. In the case ofpotential Down's syndrome patients, treatment can begin antenatally byadministering therapeutic agent to the mother or shortly after birth.

Patient selection can be done by any of the selection criteria appliedin the art of treating AD and related tauopathies. In one embodiment,the criteria are those determined by a particular clinical trial. Inanother embodiment, the criteria are those determined by an approvedtreatment regimen. A combination of inclusion and exclusion criteria maybe applied. Examples of inclusion criteria include, but are not limitedto: diagnosis of probable Alzheimer's disease based on the NINCDS/ADRDAcriteria; confirmed AD of mild to moderate degree if the MMSE score isin the range of 15 to 26; the result of the Magnetic Resonance Imagingscan (MRI) of the patient's brain is consistent with the diagnosis ofAD; presence of tau pathology in the patients' brains determined bysuitable imaging methods, e.g. by using (11)C-PBB3 or lansoprazole-basedradiopharmaceuticals; and age between 50 and 85 years.

In one embodiment, patient selection follows the method described inRollin-Sillaire et al. Reasons that prevent the inclusion of Alzheimer'sdisease patients in clinical trials, Br J Clin Pharmacol. April 2013;75(4): 1089-1097.

A number of outcome measures and primary endpoints are provided hereinfor some embodiments of how treatment is evaluated and effective amountdetermined. In one embodiment, the primary outcome measures at a giventime frame include Mean change in Alzheimer's Disease ActivityScale-Cognitive subscale 13 (ADAS-Cog13) scores and Mean change inAlzheimer's Disease Cooperative Study-Activities of Daily Living(ARCS-ADL) scores and the secondary outcome measures include Change inbiomarkers (total-tau, phosphorylated-tau, Abeta 1-42 levels) incerebral spinal fluid, Change in MRI volumetry, assessed on structuralMRI, Change in Clinical Dementia Rating (CDR-SB/CDR-GS), Change inneuropsychiatric behavior: Neuropsychiatric Inventory (NPI) total anddomain scores, Change in cognition: MMSE total score, and/or Safety:Incidence of adverse events, serious adverse events and treatmentdiscontinuations. In another embodiment, the primary end points includeany of the following: time to the occurrence of death,institutionalization, loss of ability to perform activities of dailyliving, severe dementia, slowing of the rate of progression of thedisease, ADCS-ADL, ADAS-cog score, MMSE scores, cognitive performance,plasma CSF biomarkers, ADAS-total score, Quality of life assessed byQuality of Life Alzheimer's disease scale, behavioral test scores, andthe US FDA's Clinical Dementia Rating-sum of boxes.

In one embodiment, treating Alzheimer's disease refers to decreasing orpreventing behavioral, functional, and cognitive deterioration overtime. In some embodiments, behavioral, functional, and cognitive aspectsof Alzheimer's Disease can be evaluated by any one or more of a seriesof standardized tests known to persons of ordinary skill in the artincluding, but not limited to, neuropsychological testing, theMini-Mental State Exam, Mini-cog exam, Neuropsychiatric Inventory,Blessed Roth Dementia Rating Scale, Spanish and EnglishNeuropsychological Assessment Scales (SENAS), Psychiatric BehavioralAssessment, Functional Assessment, Clock Drawing Test, Boston NamingTest, California Verbal Learning Test, Cognitive Symptoms Checklist,Continuous Performance Test, Controlled Oral Word Association Test,Cognistat, d2 Test of Attention, Delis-Kaplan Executive Function System,Dementia Rating Scale, Digit Vigilance Test, Figural Fluency Test,Finger Tapping Test, Halstead Category Test, Halstead-ReitanNeuropsychological Battery, Hooper Visual Organization Test, KaplanBaycrest Neurocognitive Assessment, Kaufman Short NeuropsychologicalAssessment, Luria-Nebraska Neuropsychological Battery, Memory AssessmentScales, Quick Neurological Screening Test, Repeatable Battery for theAssessment of Neuropsychological Status, Stroop Test, Symbol DigitModalities Test, Tactual Performance Test, Thematic Apperception Test,Tower of London, Trail Making Tests A and B, Verbal (Word) FluencyTests, and Wisconsin Card Sort Test. Additional tests for depression,anxiety, aphasia, agitation, and behavioral parameters known to personsof ordinary skill in the art are also used.

In another embodiment, the treatment of Alzheimer's disease isdetermined by the improvement, or no deterioration, or a reduction inthe rate of deterioration in at least one of the assessments selectedfrom the group consisting of Alzheimer's Disease AssessmentScale-cognitive subscale (ADAS-cog), the Clinical Dementia Rating Sum ofBoxes (CDR-sb), the Alzheimer's Disease Cooperative Study Activities ofDaily Living Scale (ARCS-ADL), the Neuropsychiatric Inventory (NPI), andthe Mini-Mental State Evaluation (MMSE). In some embodiments, thetreatment results in a reduction in the rate of deterioration inADAS-cog scores. In other embodiments, the treatment results in a medianreduction in the rate of deterioration of ADAS-cog scores of two to fivepoints.

In one embodiment, patients are identified and/or selected based on theFDA's “Guidance for Industry, Alzheimer's Disease: Developing Drugs forthe Treatment of Early Stage Disease,” available in updated form fromthe U.S. Food and Drug Administration.

In one embodiment, diagnosis of Alzheimer's disease in human subjects ismade according to criteria of the National Institute of Neurologic andCommunicative Disorders and Stroke-Alzheimer's disease and RelatedDisorders Association (NINCDS-ADRDA).

Periodic use of one or more of these tests can advise a physician orother medical professional as to the progression, or regression ofAlzheimer's Disease and related tauopathies and the need for furthertreatment. The choice of test and the determination of success oftreatment are within the expertise of medical professionals in theAlzheimer's Disease field. An improved score in one or more tests is anindication of decrease in severity of the Alzheimer's Disease in thatsubject.

Methods of Diagnosis

Because of their ability to detect pathological forms of tau, theantibodies and tau-binding fragments thereof described herein are usefulfor numerous other practical applications. Examples of such applicationsinclude, but are not limited to, pathological tau association analysis,disease predisposition screening, disease diagnosis, disease prognosis,disease progression monitoring, determining therapeutic strategies basedon an individual's type and level of pathological tau, developingtherapeutic agents based on pathological tau levels and type associatedwith a disease or likelihood of responding to a drug, stratifying apatient population for clinical trial for a treatment regimen, andpredicting the likelihood that an individual will respond to atherapeutic agent. In vitro methods for detection of the pathologicaltau proteins associated with Alzheimer's disease and related tauopathieswith the antibodies and tau-binding fragments that are disclosed hereininclude, but are not limited to, enzyme linked immunosorbent assays(ELISAs), radioimmunoassays (RIA), Western blots, immunoprecipitations,immunofluorescence, sandwich assays, and tissue arrays.

In one embodiment, the antibodies and tau-binding fragments thereofdescribed herein can be used to assess pathological tau abnormal tissuedistribution, abnormal expression during development, or expression inan abnormal condition, such as Alzheimer's disease and relatedtauopathies. Additionally, antibody detection of circulatingpathological tau can be used to identify tau turnover during treatmentof AD and related tauopathies.

In related embodiments, the invention provides a method of diagnosing orscreening a subject for the presence of Alzheimer's disease or anothertauopathy in a subject, or for determining a subjects risk fordeveloping Alzheimer's disease or another tauopathy, the methodcomprising:

-   -   a) contacting the subject, or a cell, tissue, organ, fluid, or        any other sample of the subject, with an effective amount of at        least one antibody and/or tau-binding fragment thereof as        provided herein; and    -   b) determining the presence of a complex comprising pathological        tau and the antibody and/or tau-binding fragment thereof,        wherein the presence of the complex is diagnostic of Alzheimer's        disease or another tauopathy associated with the presence of        pathological tau.

In some embodiments, the antibodies and/or tau-binding fragments thereofcan be used to detect pathological tau in vivo, ex vivo, in situ, invitro, in a bodily fluid (e.g., blood, serum, urine, plasma,cerebrospinal fluid, saliva), or in a cell lysate or supernatant inorder to evaluate the amount and pattern of expression. In oneembodiment, the antibodies and tau-binding fragments thereof are usedfor in vivo diagnostic assays, such as in vivo imaging. In some of thoseembodiments, the antibody is labeled with a radionuclide (such as ¹¹¹In,⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, or ³H) so that the cells or tissue of interestmarked by the presence of the antibody (and pathological tau) can belocalized using immunoscintiography. Other detectable labels include alabel which is observable using analytical techniques including, but notlimited to, fluorescence, chemiluminescence, electron-spin resonance,ultraviolet/visible absorbance spectroscopy, infrared spectroscopy, massspectrometry, nuclear magnetic resonance, magnetic resonance,radiometric and electrochemical methods.

In one embodiment, the antibodies and tau-binding fragments thereof canbe used in a method for evaluating the efficacy of a treatment ofAlzheimer's disease or another tauopathy. In one embodiment, the methodcomprises using one of the antibodies and tau-binding fragments thereoffor monitoring the presence of pathological tau before, throughout, andafter the treatment. In one embodiment, a decrease in the level of, ortype of, pathological tau is indicative of a positive response to thegiven treatment. In another embodiment, a lack of change or an increasein the level of, or type of, pathological tau is indicative that thetreatment should continue.

In some embodiments, a first time point can be selected prior toinitiation of a prophylaxis or treatment and a second time point can beselected at some time after initiation of the prophylaxis or treatment.Pathological tau levels can be measured in each of the samples takenfrom different time points and qualitative and/or quantitativedifferences noted. A change in the amounts of one or more of themeasured pathological tau levels from the first and second samples canbe correlated with prognosis, used to determine treatment efficacy,and/or used to determine progression of the disease in the subject.

Detection of an antibody or tau-binding fragment thereof as describedherein can be facilitated by coupling (i.e., physically linking) theantibody or tau-binding fragment thereof to a detectable substance.Detectable substances include, but are not limited to, various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵Sand ³H.

Other Uses

The antibodies and tau-binding fragments described herein can also beused to isolate the pathological tau proteins from a natural cell sourceor from recombinant host cells by standard techniques, such as affinitychromatography or immunoprecipitation. In another embodiment, theantibodies and tau-binding fragments described herein can be used toidentify other antibodies that bind to pathological tau by competitionassays.

In one embodiment, the antibodies may be used as affinity-purificationagents. In one embodiment, the antibodies or tau-binding fragmentsthereof are immobilized on a solid phase such as a SEPHADEX™ resin ormagnetic beads (e.g. of Dynal brand) or filter paper, using methods wellknown in the art. The immobilized antibody is contacted with a samplecontaining the tau (or fragment thereof) to be purified, and thereafterthe support is washed with a suitable solvent that will removesubstantially all the material in the sample except the tau protein,which is bound to the immobilized antibody. Finally, the support iswashed with another suitable solvent, such as glycine buffer, pH 5.0,which will release the tau protein from the antibody.

Also provided are kits for using antibodies and tau-binding fragmentsthereof, such as kits for detecting the presence of pathological tau ina test sample. An exemplary kit can comprise antibodies and tau-bindingfragments thereof such as a labeled or labelable antibody and a compoundor agent for detecting variant proteins in a biological sample: meansfor determining the amount, or presence/absence of variant protein inthe sample; means for comparing the amount of variant protein in thesample with a standard; and instructions for use.

In some embodiments, the invention provides a medical device comprisingan antibody or tau-binding fragment as provided herein, wherein thedevice is suitable for contacting or administering the antibody ortau-binding fragment by at least one mode selected from parenteral,subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracerebellar, intracerebroventricular, intrathecal,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravesical, intralesional, bolus, vaginal, rectal, buccal, sublingual,intranasal, and transdermal. In one embodiment, the device comprises asyringe (e.g., a pre-filled syringe). In another embodiment, the devicecomprises a patch. In another embodiment, the device comprises a pump(e.g., a mini-pump). In another embodiment, the device comprises aninhaler. In another embodiment, the device comprises a nebulizer.

In another embodiment, an article of manufacture containing materialsuseful for treatment of AD and related tauopathies is provided. Thearticle of manufacture may comprise a vial with a fixed dose of thehumanized antibody and/or tau-binding fragment thereof contained thereinand, optionally, a package insert. The vial may be formed from a varietyof materials such as glass or plastic, and may be sealed by a stopperpierceable by a syringe. For example, the vial may be a formal vitrumtype I glass vial (e.g. 20 cc vial for a certain fixed dose or 50 ccvial for another fixed dose), with DAIKYO GREY fluoro-resin laminatedstopper, and 20 mm flip top aluminum cap. The article of manufacture mayfurther include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles, andsyringes, and the like. In one embodiment, the article of manufacturecomprises two vials, wherein a first vial contains a given fixed dose ofthe humanized antibody and/or tau-binding fragment thereof, and a secondvial contains a different fixed dose of the humanized antibody and/ortau-binding fragment thereof.

The antibodies can be used to generate anti-idiotype antibodies. (see,e.g., Greenspan & Bona, FASEB J. 7(5):437-444, 1989; and Nissinoff, J.Immunol. 147:2429-2438, 1991). Such anti-idiotype antibodies can beutilized in pharmacokinetics, pharmacodynamics, biodistribution studiesas well as in studies of clinical human-anti-human antibody (HAHA)responses in individuals treated with the antibodies. For example,anti-idiotypic antibodies bind specifically the variable region ofhumanized DC8E8 antibodies and therefore can be used to detect humanizedDC8E8 antibodies in pharmacokinetic studies and help to quantifyhuman-anti-human antibody (HAHA) responses in treated individuals.

All publications, patent applications, and patents cited in thisspecification are herein incorporated by reference as if each individualpublication, patent application, or patent were specifically andindividually indicated to be incorporated by reference. In particular,all publications, patent applications, and patents cited herein areexpressly incorporated herein by reference for the purpose of describingand disclosing compositions and methodologies that might be used inconnection with the disclosed inventions.

The following abbreviations apply to the Examples provided below.

Expi293 Human Embryonic Kidney (HEK293 High density/serum free) cells

A Adenine

bp base pairs

° C. Centigrade

C Cytosine

MEM Minimal Essential Medium

DNA Deoxyribonucleic acid

ELISA Enzyme linked immuno-adsorbent assay

EC50 Concentration of antibody providing half-maximal response

EC80 Concentration of antibody providing 80% of maximal response

ECD extracellular domain

g grams

G Guanine

HRP Horseradish peroxidase

IgG Immunoglobulin-G

K G or T (IUPAC convention)

min minute

M A or C (IUPAC convention)

nm nanometer

OD optical density

PBS Phosphate Buffered Saline

PCR Polymerase chain reaction

R A or G (IUPAC convention)

RNA Ribonucleic acid

RT Room Temperature

s second

S C or G (IUPAC convention)

T Thymine

TBS Tris Buffered Saline

UV Ultra Violet

V A or C or G (IUPAC convention)

VH Immunoglobulin heavy chain variable region

VK Immunoglobulin kappa light chain variable region

W A or T (IUPAC convention)

Y C or T (IUPAC convention)

EXAMPLES Example 1: Generation of a Chimeric Version of the DC8E8Antibody

Total RNA was isolated from DC8E8 hybridoma cells using the RNeasy Miniprotocol for isolation of total RNA (Qiagen). DC8E8 RNA (3 μg) wasreverse-transcribed to produce DC8E8 cDNA using the GE Life Sciences 1ststrand cDNA synthesis kit following the manufacturer's protocol. DC8E8cDNA was amplified by PCR in 3 separate reactions as described inSection 8.3. Immunoglobulin heavy chain variable region (VH) cDNA wasPCR-amplified with heavy chain primers M13-MHV6 plus MHCmix and kappalight chain PCR primers M13-MKV5 plus MKC using the PhusionHigh-Fidelity PCR Master Mix (Thermo Scientific). The result of each PCRreaction was a single amplification product that was purified using theQIAquick PCR purification kit and sequenced in both directions using theM13-Forward (TGTAAAACGACGGCCAGT (SEQ ID NO: 158)) and M13-Reverseprimers (CAGGAAACAGCTATGACC (SEQ ID NO: 159)).

Name Sequence (5′3′) MHV6 TGTAAAACGACGGCCAGT ATGGCTGTCCTAGGGCTACTCTTCTGC(SEQ ID NO: 160) MHCG1 CAGGAAACAGCTATGACC CAGTGGATAGACAGATGGGGG(SEQ ID NO: 161) MHCG2a CAGGAAACAGCTATGACC CAGTGGATAGACCGATGGGGC(SEQ ID NO: 162) MHCG2b CAGGAAACAGCTATGACC CAGTGGATAGACTGATGGGGG(SEQ ID NO: 163) MHCG3 CAGGAAACAGCTATGACC CAAGGGATAGACAGATGGGGC(SEQ ID NO: 164)

MHV indicates primers that hybridize to the leader sequences of mouseheavy chain variable region genes, MHCG indicates primers that hybridizeto the mouse constant region genes. Italicized sequence indicates theM13 Forward or the M13 Reverse Sequencing Primer.

Name Sequence (5′→3′) MKV5 TGTAAAACGACGGCCAGTATGGATTTTCAGGTGCAGATTATCAGCTTC (SEQ D NO: 165) MKC CAGGAAACAGCTATGACCACTGGATGGTGGGAAGATGG (SEQ ID NO: 166)

MKV indicates primer that hybridizes to leader sequences of the mousekappa light chain variable region genes; MKC indicates the primer thathybridizes to the mouse kappa constant region gene. Coloured sectionindicates the M13 Forward or the M13 Reverse Sequencing Primer. Theconsensus sequence of DC8E8 VK PCR was designated DC8E8 VK (FIG. 3) andthe consensus DNA sequence of VH designated DC8E8 VH (FIG. 4). Germ Lineanalysis of the sequences shows that the Kappa Light Chain is a MurineVK8, with no somatic mutations (FIG. 5). The Heavy Chain is a MurineVH1, and shows a number of somatic mutations in both the CDR's andFrameworks (FIG. 6). The appearance of two Proline residues in Framework1 was considered to be significant. These residues may have a structuralrole outside of the 4 Å Proximity Residues.

Construction of chimeric expression vectors entailed routine cloning theamplified variable regions into IgG/kappa vectors (herein labeled pHuG1,pHuG4 and pHuK, a variety of which are commercially available). Theclones generating the correct constructs were selected and sequenced.

Expi293 (Invitrogen) suspension cells growing in Expi293 transfectionmedium and antibiotics were co-transfected with DC8E8 VH.pHuG1 and DC8E8VK.pHuK or DC8E8 VH.pHuG4 and DC8E8 VK.pHuK (50 μg DNA each) usingExpiFectamine 293 Reagent. The cells were grown in 100 ml growth mediumfor 10 days. The presence of γ1κ and γ4κ (chimeric DC8E8 antibodies) wasmeasured in the conditioned medium by routine ELISA methods.

The tau protein-binding activity of each chimeric antibody was measuredby Binding ELISA and compared to that of the original mouse DC8E8antibody. Each well of a 94-well MaxiSorp plate (Nunc) was coated with50 μL aliquots of 330 ng/mL of Tau 151-391/4R in PBS and incubatedovernight at 4° C. Wells were washed 3× with PBS-T (0.1% Tween20). Afresh plate was blocked with 250 μL of PBS/0.2% BSA/0.05% Tween20 perwell and incubated for 1 hour at RT. Wells were washed 3× with PBS-T(0.1% Tween20). 240 μL were added of antibody (diluted in PBS/0.2%BSA/0.05% Tween20 if necessary) to wells in column 1; 120 μL of buffer(PBS/0.2% BSA/0.05% Tween20) in the other wells. 120 μL were transferredfrom column 1 to the neighboring wells in column 2. The procedure wasontinued to column 12 with a series of 2-fold dilutions of theexperimental samples. 100 μL per well were transferred from the dilutionplate to the experimental plate. Plates were incubated for 1 hour at RT.Wells were washed 3× with PBS-T. The goat anti-human Fc peroxidaseconjugate was diluted 10000-fold (or anti-mouse at 10000-fold dilution)in PBS/0.2% BSA0.05% Tween20 and 100 μL added to each well. Plates wereincubated 1 hour at RT and repeated washing step. 150 μL of substrate(K-Blue) were added per well and incubated for 150 minutes at RT. Thereaction was stopped by adding 50 μl of RED STOP solution to each well.The optical density was read at 650 nm. Both chimeric antibodies boundTau 151-391/4R with comparable EC₅₀ values, comparable with the murineDC8E8 antibody (FIG. 7). The sequence was used to design the humanizedversion of the DC8E8 antibody.

Example 2: Humanized Variants of Mouse DC8E8

The immunoglobulin sequence M65092 was chosen as the human donorcandidate for the humanized heavy chain framework (FW) due to its highersequence identity and similarity to the DC8E8 variable heavy chainregion (VH). The sequence alignment of these two variable regions can befound in FIG. 8. The next step was to graft CDR1, 2, and 3 from DC8E8 VHinto the acceptor FW of M65092. The human residues at Kabat positions 9,21, 27, 28, 30, 38, 48, 67, 68, 70 and 95 are not conserved in thewild-type variable heavy chain region (RHA) of M65092. Due to theirposition (within 4 Å of a Kabat CDR using the program Discovery Studio(Accelrys), except for Pro at positions 9 and 21) these residues weretested for their importance in tau binding. This step is one of the mostunpredictable procedures in the humanization of monoclonal antibodies,and necessitates the identification of critical framework residues fromthe parent antibody that need to be retained in order to substantiallyretain the binding properties of the parent antibody while at the sametime minimizing the potential immunogenicity of the resultant humanizedantibody. Each of these non-conserved residues was back-mutated to theDC8E8 mouse equivalent residue, resulting in the various recombinantvariable heavy chain regions RHA through RHM. (FIG. 8).

In order to humanize the light chain a human kappa (light) chain wasidentified in a similar process to that of the heavy chain. Initialanalysis found several potential donor candidates, but all these provedto be Human VK4, which show poor expression. Extending the analysis toinclude CDR1 with one less residue resulted in a single candidate,X72449, which showed a higher degree of sequence homology to the murineantibody than the VK4 candidates. The sequence of DC8E8 variable kappalight chain (VK) was aligned with the variable kappa light chain ofX72449. FIG. 9. RKA shows the recombinant variable light chain havingDC8E8's CDRs grafted onto the X72449 framework. In an alternativevariant, RKB, DC8E8's CDRs grafted onto the X72449 framework and Kabatresidue 5 was back mutated to the corresponding DC8E8 light chain mouseresidue.

The genes for RHA through RHM, RKA, and RKB were synthesized byGenScript and/or PCR mutagenesis. The genes were codon optimized bysilent mutagenesis to use codons preferentially utilized by human cells,using software algorithms proprietary to GenScript. Each of the RHAthrough RHM genes was then inserted into human IgG1 and IgG4 expressionvectors, using methods typically done in the art. RKA and RKB genes wereinserted into a K light chain expression vector in the same manner.Multiple examples of such expression vectors are well known andcommercially available to one of ordinary skill in the art. Clones weresequenced and expression plasmid DNA prepared using QIAGEN PlasmidMiniprep/Maxiprep Kits. Expression plasmid preparations encoding alldifferent humanized and chimeric VH and VK sequences were used totransfect Expi293 cells using Invitrogen's Expi293 expression systemkit, cultured for 10 days in serum free media, whereupon the conditionmedium containing each secreted antibody was harvested. When desired,the concentrations of IgG1k and IgG4k antibodies in Expi293 conditionedmedia were measured by ELISA using routine methods of antibodyquantification.

Example 3: Properties of the Humanized Versions of DC8E8

Tau Protein Binding by DC8E8 Antibodies

Binding activity to Tau protein (151-139/4R) was measured by BindingELISA, as described in Example 5. The data shown in FIG. 10 show thebinding potency of the initial versions of the humanized DC8E8. Althoughall versions bound to the Tau protein, of this initial set, thoseantibodies containing the RHB version of the heavy chain appeared tobind better. This version of the heavy chain contains back-mutations tomurine residues at all of Kabat positions 9, 21, 27, 28, 30, 38, 48, 67,68, 70 and 95, indicating that one or more of these residues isimportant for retaining full antibody binding activity.

Further versions of the humanized heavy chain were synthesized, eachwith a single back-mutation, and were tested for binding by ELISA.Results for the IgG4 versions are shown and summarized in FIGS. 11, 12,and 13. Versions of the antibody having RHB, RHD and RHE wereconsistently better, with either of the humanized light chains (RKA andRKB) or chimeric light chain (cDC8E8). As the RHD and RHE versionscontain only a single murine back-mutation, as opposed to the 11 murineback-mutations in version RHB, these two versions were chosen aspossible lead candidates, in conjunction with the RKA light chainversion (no murine back-mutations). It was surprising that theseback-mutants would have been sufficient to restore binding affinity tothe humanized antibodies. Combining RHD and RHE mutations (version RHM)did not result in a marked improvement in pathological tau-bindingproperties, relative to each individual mutation.

In light of the results described above, a heavy chain version (RHM)containing both murine back-mutations present in RHD and RHE wasgenerated by site-directed mutagenesis. FIG. 14 shows the Tau protein151-391/4R binding of the RHM versions of the humanized DC8E8 antibody,and indicates that the affinity for the protein is no better than thelead candidate, AX004 (RHD/RKA).

Thermo Stability of Humanized Candidate Antibodies to High Temperatures

The aim of this experiment is to compare the thermo stability of thehumanized antibodies. When subjected to higher temperatures, varyingfrom 30° to 85° C. for 10 minutes, cooled to 4° C. and used in anBinding ELISA assay at the EC80 concentration of each candidate. All theantibodies tested appear equally stable (FIG. 15), all becoming inactiveonly at 70° C., but without any untoward effect on tau-bindingproperties before then.

Determination of Humanized Candidate Antibodies Tm

In order to determine the melting temperature of the lead antibodiesAX004 (DA), AX005 (EA), AX016 (DB) and AX017 (DE), the humanized,chimeric, and mouse antibodies were purified in a 2-step affinitychromatography and gel filtration system (as described in Example 4) andtested in a thermal shift. This thermal shift assay is a microplatebinding assay for monitoring thermal melting curves. The method uses anenvironmentally sensitive dye SYPRO Orange, whose protein bindingproperties increase with denaturation of the protein. Therefore, uponheat-induced denaturation of the analyzed protein the binding of the dyeincreases and reaches maximum when the protein is fullydenatured/unfolded. The fluorescence intensity plotted as a function oftemperature thus generates a typical sigmoid curve, where inflectionpoint (half maximal intensity) corresponds to the melting temperature(Tm) of the proteinSamples were incubated with a fluorescent dye (SyproOrange) for 71 cycles with 1° C. increase per cycle in a qPCR thermalcycler. Tm for the chimeric and the four humanized antibodies areindicated in FIG. 16 and confirm the results obtained in the thermostability assay: all of the candidate antibodies have very similar Tms(69-70° C.) and are similar to those of the chimeric antibody (70° C.)but slightly lower than that for the mouse (73° C.) antibody.

Affinity of Humanized Candidate Antibodies

Antibody affinity determination was carried out by SPR analysis using aBiacore T200. Anti-human IgG (GE Healthcare, cat. no. BR-1008-39) oranti-murine IgG (GE Healthcare, cat. no. BR-1008-38) was covalentlyimmobilized to a CM5 chip (Cat. no. BR-1005-30) to the point ofsaturation and in accordance with the manufacturer's instructions. Theantibodies mouse DC8E8, chimeric DC8E8, AX004, AX005, AX016 and AX017were diluted in HBS-EP buffer [0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mMEDTA, 0.005% v/v Surfactant P20, GE HEalthcare] and immobilized byaffinity binding to a level of between 144 and 177 RU. Recombinantmonomeric tau 151-391/4R (in PBS saturated with Argon) was diluted inHBS-EP, in concentrations ranging from 20 nM to 0.312 nM in doublingdilutions and then injected at a flow rate of 100 μL/min. Associationtime was set at 150 sec and disassociation time 300 sec. Tauconcentrations were chosen empirically in order to obtain curvatureduring the association phase. The kinetics of binding/dissociation wasinitially analyzed according to the 1:1 interaction model using BIAcoreT200 evaluation software package 2.0.

Sensograms are shown in FIGS. 17-22. Unfortunately, due to the biphasicnature of the dissociation phase, and some evidence of extra bindingduring the association phase, it was not possible to analyze thekinetics. The data shown was obtained using a Steady State Analysis.Both the murine and chimeric DC8E8 show an affinity of approximately 10nM. The lead humanized antibodies show similar affinities, ranging from9 mM (RHD/RKB) to approximately 16 nM (RHE/RKA), summarized in FIG. 23.These results suggest that the humanization has successfully retainedthe binding activity within the desired parameters.

Aggregation of Humanized Candidate Antibodies

Aggregation is considered a serious problem in drug development, asaggregation may lower yield during production, limit shelf-life andresult in reduced potency in patients due to increased immunogenicity.Antibody samples were injected at 0.4 mL/min into a size exclusioncolumn in an HPLC system and analyzed by multi-angle light scattering todetermine the absolute molar masses and check for aggregation (see FIGS.24A, B, C, and D). All variants show no signs of aggregation with anaverage molecular weight ranging from 160-169.8 kDa, which is theexpected range for an IgG monomer in this analysis setup. All samplesare monodispersed (Mw/Mn<1.05). The mass recoveries are between99.6-99.9% (calculated mass over injected mass), which indicates goodprotein recovery and that the samples do not seem to stick to the columnor contain insoluble aggregates, which would be retained by the guardcolumn. Overall the data suggest there are no aggregation concerns inany of the anti-DC8E8 antibody samples analyzed.

Solubility of Humanized Candidate Antibodies

The purified candidate antibodies were concentrated using solventabsorption concentrators (MWCO 7500 kDa) and the concentration measuredat timed intervals. All of the samples were concentrated to between 35and 41 mg/ml without apparent precipitation, and tested in Binding ELISAthat showed that none had lost binding potency to Tau Peptide (FIGS. 25and 26). The data suggest that the antibodies are not prone toprecipitation at concentrations up to at least 35 mg/ml.

Freeze/Thaw Stress Analysis of Candidate Antibodies

Samples of the purified candidate antibodies were subjected to 10 cyclesof 15 minutes at −80° C. followed by thawing for 15 minutes at RoomTemperature. Samples were then analyzed by SEC-MALS to analyze foraggregation (FIG. 27). The data suggests that freeze/thaw does notinduce aggregation for all four antibodies tested. Overall, the datasuggest there are no aggregation concerns in any of the anti-DC8E8antibody samples analyzed.

Heat-Induced Stress Analysis of Candidate Antibodies

Samples of the purified candidate antibodies were heat exposed at a)Room Temperature, b) 37° C. and c) 50° C. for 20 days. Samples were thenanalyzed by SEC-MALS to analyze for aggregation (FIG. 28). Overall, thedata suggest there are no aggregation concerns in any of the anti-DC8E8antibody samples analyzed.

Example 4: Preparation of Recombinant Full-Length Tau Isoform 2N4R andMisdisordered Tau 151-391/4R

Recombinant tau proteins were generated from clone τ40 (Goedert, 1989),which was subcloned into the expression plasmid pET-17b (Novagen) andexpressed in bacteria. The misdisordered tau 151-391/4R and all taupeptides are numbered according to the longest human tau isoform 2N4R,which is 441 amino acids in length and thus is also called tau441(D'Souza, 2005). Production of tau proteins involved the followingsteps: a) expression of tau in bacteria; b) tau purification by ionexchange chromatography; c) tau purification by gel-filtration; d)concentration and storage of isolated tau;

a) Bacterial expression of human full-length tau 2N4R and misdisorderedtau151-391/4R: expression plasmids were transformed into Escherichiacoli production strain BL21(DE3). Bacterial cells containing theappropriate expression plasmid were cultivated and induced as describedin “Molecular Cloning: A Laboratory Manual” by Sambrook and Russell(2001). A single colony of BL21(DE3) bacteria, transformed with pET-17bplasmid driving expression of a tau protein or its fragment, were grownat 37° C. in 500 ml of Luria broth medium with 100 μg/ml ampicillin at300 rpm and induced by the addition ofisopropyl-β-D-1-thiogalactopyranoside (IPTG) to a final concentration of0.4 mM. After further incubation at 37° C. for 3 hours, bacteria werecollected by centrifugation at 3,000×g for 15 min at 4° C.

b) Cation-exchange chromatography purifications of the basic and neutraltau proteins (full-length tau isoforms and tau 151-391/4R) were doneessentially as previously described (Krajciova et al., 2008). Afterexpression, the bacterial pellets were resuspended in 10 ml of lysisbuffer (50 mM 1,4-piperazinediethanesulfonic acid (PIPES) pH 6.9, 50 mMsodium chloride (NaCl), 1 mM ethylenediaminetetraacetic acid (EDTA), 5mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 5%(v/v) glycerol), quickly frozen in liquid nitrogen, and stored at −80°C. until used for purification of tau proteins. For tau proteinpurification, the frozen bacterial suspensions were quickly thawed andplaced on ice. Bacterial cell walls were broken by sonication on ice byusing Sonopuls HD 2200, tip TT-13 (Bandelin, Germany) set to 50% dutycycle, 50 W power output, 6 times for 30 s with 30 s pauses. The lysateswere clarified by centrifugation (21,000×g for 15 min at 4° C.) and thesupernates were filtered through a 0.45 μm membrane filter. Large-scalepurification of the recombinant tau proteins was done at 6° C. using anÄKTA-FPLC workstation (Amersham Biosciences, Sweden). The filteredlysates were loaded at a 3 ml/min flow rate onto a 5-ml HiTr ap SP HPcolumn (GE Healthcare, Uppsala, Sweden) equilibrated with the lysisbuffer, and washed extensively with 60 ml of the lysis buffer until thebaseline at 280 nm became stable. Bound tau proteins were eluted by agradient (0-30% within 15 ml) of Buffer B (lysis buffer supplementedwith 1 M NaCl). Individual 1 ml fractions were collected and analyzed bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Toremove nucleic acids, which copurify with positively charged tauproteins, the fractions containing tau protein were pooled and purifiedby a second cation-exchange chromatography step, using a 5-ml HiTrap SPHP column (GE Healthcare, Uppsala, Sweden) with a less steep gradient ofBuffer B (0-30% in 45 ml).

c) In the final gel-filtration step of purification (the same for alltau proteins), pooled tau protein fractions obtained by ion exchangechromatography, were injected onto a gel-filtration column (HiLoad 26/60Superdex 200 prep grade column, GE Healthcare) at 3 ml/min in eitherPIPES or Histidine lysis buffer for basic/neutral or acidic tauproteins, respectively, supplemented with 100 mM NaCl, Eluted tauproteins were pooled.

d) For tau protein concentration after gel-filtration purification,pooled fractions were diluted with 1.5 volumes of 2.5% glycerol, andloaded again on a HiTrap SP HP column (basic and neutral tau proteins)or on a HiTrap Q HP column (acidic tau proteins). The concentratedrecombinant tau protein was then eluted from the column with a 1 M NaClstep gradient. Finally, the buffer was exchanged to phosphate-bufferedsaline (PBS, 8.09 mM disodium phosphate (Na₂HPO₄), 1.47 mM potassiumdihydrogen phosphate (KH₂PO₄), 136.89 mM NaCl, 2.7 mM potassiumchloride(KCl)) saturated with argon, using a 5 ml HiTrap Desalting column (GEHealthcare). Protein quantitation of purified samples was done usingbicinchoninic acid (BCA) quantitation kits (Pierce, USA), with bovineserum albumin (BSA) as a standard. Tau proteins were aliquoted intoworking aliquots, snap-frozen in liquid nitrogen, and stored at −70° C.

Example 5: Properties of Chimeric DC8E8

a/ Chimeric DC8E8 Displays Higher Affinity for Misdisordered Tau151-391/4R than for Full Length Tau 2N4R.

The discriminatory capacity of chimeric DC8E8 between immunoreactivitywith pathological tau and physiological tau was determined by ELISA andby surface plasmon resonance (SPR).

For ELISA and SPR experiments, mouse DC8E8 was purified from serum-freehybridoma supernate on a Protein G affinity column, as follows. Thehybridoma supernate was adjusted to pH 7.5 by adding 0.2 volume of PBSand checking with pH paper strip (if necessary, pH is further adjustedby adding 4 M NaOH), the solution was precleared by centrifugation(20,000×g, 4° C., 10 minutes), filtered through a 0.45 μm membranefilter, and loaded onto a 5 ml Protein G Sepharose column (at a flowrate of 1-0.5 ml/min). DC8E8 was eluted from the column with 0.1 MGlycine-HCl, pH 2.7. Eluted fractions were immediately neutralized with1M Tris-HCl pH 9.0. Pooled fractions were dialyzed against PBS,concentrated by ultrafiltration, and stored at −70° C. The concentrationof the antibody was determined by measuring absorbance at 280 nm, usingthe formula c(mg/ml)=A280 nm/1.43.

The chimeric DC8E8 was expressed in Expi293 cells and purified fromserum-free conditioned media by affinity chromatography andsize-exclusion chromatography as described above for mouse DC8E8 with afew modifications. The cell culture medium was adjusted to pH 7.5,precleared by centrifugation, filtered through a 0.45 μm membranefilter, and loaded onto a 1 ml HiTrap MabSelect SuRe Protein A column,using Dulbecco PBS as binding and washing buffer. Chimeric DC8E8 mAb waseluted from the column with 0.1M sodium citrate pH 2.5 supplemented with150 mM NaCl. Eluted fractions were immediately neutralized with 1MTris-HCl pH 9.0. Pooled fractions were polished by size exclusionchromatography on the HiLoad 16/600 Superdex 200 pg column, Dulbecco PBSas running buffer. The concentration of the antibody was determined bymeasuring absorbance at 280 nm, using the formula c(mg/ml)=A280 nm/1.37.

For direct ELISA assays, mis-disordered tau151-391/4R or full length tau2N4R were immobilized on ELISA plates (Nuns, MediSorp) at 5 μg/ml inPBS, 50 μl/well, and incubated overnight at 37° C. After blocking withPBS-0.05% Tween 20 (1 hr at 20-25° C.) to reduce nonspecific binding,the plates were incubated with 50 μl/well of three-fold serial antibodydilutions (concentration range of 10000 ng/ml-0.17 ng/ml) in theblocking buffer (PBS, 0.05% Tween 20) for 1 hr at 37° C. Afterincubation and washing, peroxidase-conjugated secondary antibody(anti-human Ig, Pierce, ThermoScientific) was diluted 1:4000 inPBS-Tween buffer and applied to the wells (50 μ/well) for 1 hr at 37° C.After washing, the reaction was developed for 20 min with colorburstblue solution (50 μ/well) as a peroxidase substrate and stopped with 50μl of 2 M H₂SO₄. Absorbance was measured at 450 nm using a MultiscanMCC/340 ELISA reader (Labsystems). Readouts with an absorbance value ofat least twice the value of the negative controls (PBS) were consideredpositive.

The analysis showed that chimeric antibody DC8E8 was able todiscriminate between the misdisordered tau 151-391/4R and physiologicaltau 2N4R (FIG. 29A). Chimeric DC8E8 recognized the physiological tau2N4R to a much lesser extent than it recognized thepathological/misdisordered tau 151-391/4R. Importantly, theimmunoreactivity of chimeric DC8E8 was comparable with immunoreactivityof mouse DC8E8 (FIG. 29B). Both chimeric and mouse DC8E8 bound analyzedtau proteins (pathological and physiological tau) with similar EC₅₀values as indicated in FIG. 29C. Together, these findings suggest thatbinding properties of chimeric DC8E8 are comparable with bindingproperties of original mouse DC8E8.

Surface plasmon resonance (SPR) is used for the detection andquantification of protein binding and for determination of thethermodynamic parameters of protein complexes (e.g., antibody-antigencomplexes) by direct monitoring of the binding event in real time. Thistechnology is routinely used to characterize both diagnostic andtherapeutic antibodies (See, e.g., Karlsson and Larsson, AffinityMeasurement Using Surface Plasmon Resonance, in Methods in MolecularBiology, Vol. 248: Antibody Engineering: Methods and Protocols. Editedby: B. K. C. Lo© Humana Press Inc., Totowa, N.J., (2008)).

A BIACORE3000 instrument with a CM5 sensor chip (Biacore AB, Uppsala)was used for the SPR assays. Amine-coupling reagents (EDC, NHS,ethanolamine pH 8.5), P20 detergent, and 10 mM sodium acetate pH 5.0were obtained from Biacore AB. These experiments were done at 25° C. inPBS pH 7.4 with 0.005% of P20 (PBS-P) as the running buffer. Forchimeric DC8E8, goat anti-human-Fc polyclonal antibody (SIGMA, Cat. no.I2136) was coupled at pH 5.0 via primary amines simultaneously in twoflow cells to 5,000 RU (response units), one of which was used forreference measurement. For mouse DC8E8, analytical flow cell was coatedwith polyclonal anti-mouse antibody (No. Z 0420; DakoCytomation,Glostrup, Denmark).

In each analysis cycle, purified chimeric DC8E8 and mouseDC8E8 wereseparately captured in the analytical flow cell to reach animmobilization level of 200-250 RU. For determinations of equilibriumassociation binding constant (K_(A)) as well as for the determination ofthe kinetic rate constants (kON and kOFF), two-fold serial dilutions ofeither tau151-391/4R and physiological tau 2N4R (against which DC8E8affinity was tested), or PBS-P as a control, were injected at a flowrate of 100 μl/min over the sensor chip. Kinetic binding data weredouble referenced as described by Myszka (1999) and fitted by BIAevaluation software 4.1 (Biacore AB) separately to obtain dissociationrate constant and association rate constant. Equilibrium associationconstant K_(A) was obtained as a ratio of association and dissociationrate constants.

In order to quantify chimeric DC8E8's affinity for tested tau proteins,the association equilibrium binding constants (K_(A)) were determinedfor DC8E8 binding to the physiological, four repeat tau protein isoform2N4R as well as to pathological, misdisordered tau 151-391/4R. Both tauproteins used for SPR were prepared as described in Example 4. ChimericDC8E8 antibody discriminates between misdisordered tau151-391/4R proteinand the physiological tau protein 2N4R (FIG. 30). The extent ofdiscriminatory potency of chimeric DC8E8 is even slightly higher thanthat of the original mouse DC8E8 antibody. These results confirmed: (1)the specificity of humanized DC8E8 for the misdisordered form of tau,and (2) the selectivity of DC8E8 for misdisordered tau (i.e., disease orpathological tau) over the full-length tau (i.e., normal orphysiological tau).

b/ Chimeric DC8E8 Binds Tau Peptides each Carrying One of the Four DC8E8Epitopes in the Repeat Regions of Microtubule-Binding Domain of ProteinTau

Previous results showed that mouse DC8E8 has four binding sites orepitopes (267-273, 298-304, 329-335 and 361-367) on human tau, each ofwhich is separately located within one of the repeats in themicrotubule-binding domain of tau (WO/2013/041962, Kontsekova et al.,2014). With the aim to test the capability of chimeric DC8E8 to bind toany one of the four epitopes, tau peptides 256-285, 282-311, 314-342,352-380 were synthetized by EZBiolabs (USA) with purity higher than 95%.Each of the peptides encompassed one of the four separate DC8E8epitopes. Binding activity of chimeric DC8E8 to the tau peptides wasmeasured by direct ELISA, as described in Example 5 a/. Chimeric DC8E8bound all tested MTBRs-derived peptides (FIG. 31A), similarly tooriginal mouse DC8E8 (FIG. 31B). Importantly, the highestimmunoreactivity showed chimeric and mouse DC8E8 to peptide 282-311,which was derived from MTBRII and which comprises DC8E8 epitope withinposition 298-304. The immunoreactivity of chimeric antibody toadditional tested peptides (256-285, 314-342, 352-380) was even slightlyhigher, than that of original mouse DC8E8, as indicate EC₅₀ values (FIG.31C).

c/ Chimeric DC8E8 Inhibits Pathological Tau-Tau Interaction

An in vitro tau fibrillization assay was used to determine whetherchimeric antibody had an inhibitory effect on pathological tau-tauinteractions. The assay is based on an intrinsic property of tauproteins, namely their ability to undergo a conformational change uponinteraction with polyanions, such as the sulfated glycosaminoglycanheparin. This altered conformation on one tau molecule further leads toits pathological interactions with another tau molecule, stabilizationof the tau-tau complex through formation of cross-β sheet structures inthe microtubule binding regions of the interacting tau molecules, and,lastly, formation of Alzheimer's disease-like paired helical filaments(PHFs) (Skrabana, R., Sevcik, I., Novak, M. (2006). Intrinsicallydisordered proteins in the neurodegenerative processes: formation of tauprotein paired helical filaments and their analysis. Cell Mol Neurobiol26, 1085-1097). The formation of the beta-sheet-rich structures can bedetected by fluorescent dyes, like Thioflavin T (Friedhoff P, SchneiderA, Mandelkow E M, Mandelkow E. Rapid assembly of Alzheimer-like pairedhelical filaments from microtubule-associated protein tau monitored byfluorescence in solution. Biochemistry 37(28):10223-30 (1998)).

For in vitro tau fibrillization assay, mouse and chimeric DC8E8 werepurified by methods described hi Example 5. The assay to measure theeffect of chimeric DC8E8 on pathological tau-tau interactions was setupin PBS (filtered through a 0.2 μm filter) containing: 10 μM (finalconcentration) of the misdisordered tau 151-391/4R, 10 μM heparin(Heparin sodium salt from porcine intestinal mucosa, ≥150 IU/mg, drybasis, average molecular weight 6000 Da, from SIGMA); and 12.5 μM (finalconcentration) Thioflavin T. Each reaction (50 μl final volume) wasincubated for 20 h at 37° C. in sealed black solid polystyrene plates(384 wells, Greiner BioOne). Thioflavin T fluorescence was measuredusing a fluorescence reader (Fluoroskan Ascent FL (Labsystems)), withexcitation wavelength of 450 nm, emission at 510 nm, and 200 msmeasurement time. For determination of the inhibitory activity ofchimeric DC8E8 on pathological tau-tau interactions, purified chimericDC8E8 was added to the reaction mix at 10 μM final concentration, priorto the incubation at 37° C. The amount of conformationally altered andfibrilized tau was measured by Thioflavin T fluorescence in the absence(“no antibody”) and in the presence of the chimeric antibody (FIG. 32).Both chimeric DC8E8 and mouse DC8E8, added at 10 μM final concentration,prevented the pathological conformational change and fibrillization ofthe misdisordered tau protein, Chimeric DC8E8 reduced the amount offibrillized pathological tau forms to less than 5.9%, mouse DC8E8reduced the amount of fibrillized pathological tau forms to less than4.7%. The data shows that chimeric antibody prevented the pathologicalconformational change and fibrillization of misdisordered tau proteinwith the comparable capacity as that of parental DC8E8.

Example 6: Properties of the Humanized Versions of DC8E8

a/ Humanized Versions of DC8E8 Exhibit Higher Affinity for MisdisorderedTau 151-391/4R than for Full Length Tau 2N4R.

To assess the discriminatory capacity of humanized versions of DC8E8between immunoreactivity with pathological and physiological tauproteins, ELISA and SPR were used, as described in Example 5. Allhumanized variants of DC8E8, namely AX004, AX005, AX016, AX017 (in bothisotypes IgG4 and IgG1), were purified according to the method forpurification of chimeric DC8E8 described in Example 5.

The ELISA showed that all humanized leads AX004, AX005, AX016 and AX017(of both IgG4 and IgG1 isotypes) were able to recognize pathologicaltau151-391/4R (FIG. 33A-D; FIG. 34A-D). Importantly, the binding of eachhumanized DC8E8 variant was higher for pathological tau 151-391/4R thanfor the physiological tau 2N4R. However, the leads containing the RHEversion of heavy chain (AX005 and AX017) appeared to bind thephysiological tau weaker than leads AX004 and AX016 containing the RHDversion of heavy chain (FIG. 33F; FIG. 34E). Although AX005 and AX017bind the physiological tau weaker, the extent of discriminatory potencyof humanized leads AX004, AX005, AX016 and AX017 is similar to that ofchimeric DC8E8 antibody, as indicated by EC₅₀ values. Altogether,binding properties of the tested humanized antibodies are comparablewith binding properties of chimeric antibody (and parental mouse DC8E8,FIG. 29).

For SPR experiments performed as described above for chimeric DC8E8, thehumanized variants of DC8E8 monoclonal antibody and chimeric DC8E8 wereused. In each analysis cycle, purified humanized version of DC8E8 wascaptured in the analytical flow cell to reach the immobilization levelsof 200-250 RU. In order to quantify humanized DC8E8's affinity for eachof the tested tau proteins, the association equilibrium bindingconstants (KA) were determined for the antibodies binding to thephysiological, four repeat tau protein isoform 2N4R as well as topathological misdisordered tau151-391/4R. All tau proteins used for SPRwere prepared according to Example 4. The affinity of each humanizedDC8E8 variant was higher for misdisordered tau 151-391/4R then for thefull-length tau 2N4R. (FIGS. 35A, B). These results confirmed: (1) thespecificity of humanized DC8E8 for the misdisordered form of tau, and(2) the selectivity of DC8E8 for misdisordered tau (i.e., disease orpathological tau) over the full-length tau (i.e., normal orphysiological tau).

b/ Humanized Versions of DC8E8 Bind Tau Peptides each Carrying One ofthe Four DC8E8 Epitopes in the Microtubule-Binding Domain of Tau

The aim of this experiment was to determine the capability of thehumanized leads of DC8E8 (AX004, AX005, AX016, AX017, isotype IgG4 andisotype IgG1), to bind tau peptides 256-285, 282-311, 314-342, 352-380.Each of these peptides encompassed one of the four separate DC8E8epitopes in the microtubule-binding repeats (MTBRs) of tau. Bindingactivity of humanized leads of DC8E8 to the tau peptides was measured byELISA, as described in Example 5. Each of the tested humanizedantibodies bound all MTBRs-derived peptides (FIG. 36A-D; FIG. 37A-D),similarly to chimeric DC8E8 (FIG. 36E). This was true for both IgG1 andIgG4 isotype versions. The humanized candidate antibodies showed thehighest immunoreactivity to peptide 282-311, which was derived fromMTBRII and which comprises DC8E8 epitope within positions 298-304, asindicated by EC₅₀ values (FIG. 36F; FIG. 37E). The leads containing theRHE version of heavy chain (AX005 and AX017) bind the other testedpeptides (256-285, 314-342, 352-380) weaker than the leads AX004 andAX016 containing the RHD version of the heavy chain (FIG. 36F; FIG.37E). Overall the data suggest that the immunoreactivity of humanizedantibody AX004 and AX016 to the tested peptides is similar to that ofchimeric DC8E8, as indicated by EC₅₀ values (FIG. 36F).

c/ Humanized Versions of DC8E8 are Capable of Inhibiting PathologicalTau-Tau Interaction

In order to determine the effect of humanized candidate antibodies(namely AX004, AX005, AX016, AX017, isotype IgG4 and isotype IgG1) ontau fibrillization and on the formation of tau aggregates, in vitro taufibrillization assay was performed (as described in Example 5). Allhumanized leads of DC8E8 were purified as described in Example 5.Pathological tau protein 151-391/4R used for fibrillization assay wasprepared according to Example 4.

For determining the inhibitory activity of humanized candidateantibodies on pathological tau-tau interactions, purified humanizedleads AX004, AX005, AX016, AX017 (isotype IgG4 and isotype IgG1) wereseparately added to the reaction mix at 10 μM final concentration, priorto the incubation at 37° C. The amount of conformationally altered andfibrillized tau was measured by Thioflavin T fluorescence in the absence(“no antibody”) and in the presence of the tested antibodies. Resultsshown, that all of the humanized antibodies, added at 10 μM finalconcentration, prevented the pathological conformational change andfibrillization of the misdisordered tau protein (FIGS. 38A, B). Isotypeof the antibodies did not influence the inhibitory potential ofhumanized antibodies. Humanized leads AX004, AX005, AX016 and AX017,both IgG4 and IgG1 isotype versions, reduced the amount of fibrillizedpathological tau forms to less than 3%. The data suggest that humanizedantibodies prevented the pathological conformational change andfibrillization of misdisordered tau protein with the comparable capacityas that of the original mouse DC8E8.

Example 7: Chimeric DC8E8 and Humanized Versions of DC8E8 RecognizePathology in Human Alzheimer's Disease Brain and Other Tauopaties

Human brain tissue samples (on paraffin blocks) were obtained from theNetherlands brain bank. The blocks were cut on a microtome.Paraffin-sections (8 μm) of the hippocampus-entorhinal cortex fromAlzheimer's disease brain (Braak's stage VI) and FTDP17 (R406Wmutation), nucleus caudatus from corticobasal degeneration andprogressive supranuclear palsy were used for the study. The sectionswere treated with cold (+4° C.) 98% formic acid for 1 min followed byheat treatment in the pressure cooker (2100 Retriever) for 20 min at121° C. The tissue sections were incubated in blocking solution (Sectionblock, Aptum) for 10 min. at room temperature and then overnight withprimary mouse antibody DC8E8 (1:200), human antibodies AX004, AX005,AX016, AX017 and chimeric DC8E8 (all 1:1000). Subsequently, the sectionswere incubated with a biotinylated secondary antibody (Vectastain EliteABC Kit, Vector Laboratories) at room temperature for an hour and thenreacted with avidin-biotin peroxidase-complex for 60 minutes (VectastainElite ABC Kit, Vector Laboratories), both at room temperature (25° C.).The immunoreaction was visualized with peroxidase substrate kit (VectorVIP, Vector laboratories, Ca, USA) and counterstained with methyl green(Vector Laboratories). The assessment of immunoreactivity was carriedout under light microscopy at ×100-400 magnification. The morphologicaldetails of tau-immunopositive lesions were defined based on the cellularlocalization and the pattern of staining. Digital images were takenusing an Olympus BX51 microscope equipped with an Olympus DP50 digitalcamera (Olympus Optical Co., Ltd., Tokyo, Japan).

Immunohistochemical staining of human brains of Alzheimer's disease,progressive supranuclear palsy, corticobasal degeneration and FTDP-17patients revealed that chimeric DC8E8 antibody displayed the samestaining pattern as mouse DC8E8 (FIGS. 39-46, A,B). We compared theimmunohistochemical staining of chimeric antibody with humanized DC8E8antibodies—AX004, AX005, AX016 and AX017. In general, humanizedantibodies AX004 and AX016 (IgG1 and IgG4) displayed the very similarstaining pattern as mouse or chimeric DC8E8. Humanized antibodies AX004and AX016 recognized extensive number of neurofibrillary tangles,neuropil threads and neuritic plaques in human AD brain (FIGS. 39C,E;40C,E). In human FTDP-17 case bearing mutation on tau protein at R406W,AX004 and AX016 immunolabeled neurofibrillary tangles and neuropilthreads in entorhinal and temporal cortex (FIGS. 41C,E; 42C,E). Incorticobasal degeneration, AX004 and AX016 stained glial tau pathologyin nucleus caudatus (FIGS. 43C,E; 44C,E). In progressive supranuclearpalsy, AX004 and AX016 recognized high number of oligodendroglial coiledbodies and astrocytic plaques (FIGS. 45C,E; 46C,E). In contrast AX005and AX017 displayed reduced immunostaining in Alzheimer's disease (FIGS.39D,F; 40D,F), in FTDP-17 (FIGS. 41D,F), in corticobasal degeneration(FIGS. 43D,F; 44D,F) and in progressive supranuclear palsy (FIGS. 45D,F;46D,F). Interestingly, isotype IgG4 of AX005 and AX017 did not stainpathological structures in FTDP17 (FIGS. 42D,F). In summary, humanizedantibodies AX004 and AX016 have the same staining pattern as DC8E8.

We claim:
 1. A humanized anti-tau antibody, or a tau-binding fragmentthereof, wherein said antibody or binding fragment comprises: a heavychain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of SEQ IDNOs. 1, 2, and 3, respectively, and a framework from humanimmunoglobulin M65092 (SEQ ID NO. 71); a light chain variable regioncomprising CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NOs. 4, 5, and 6,respectively, and a framework from human immunoglobulin X72449 (SEQ IDNO. 65); and heavy chain and light chain constant regions each from ahuman immunoglobulin; and wherein said heavy chain framework has beensubstituted at one or more positions selected from 9, 21, 27, 28, 30,38, 48, 67, 68, 70, and 95; said light chain framework has either notbeen substituted or has been substituted at position 5; and wherein saidpositions are according to Kabat.
 2. The antibody or binding fragment ofclaim 1, wherein heavy chain position 9 is occupied by P, position 21 isoccupied by P, position 27 is occupied by Y, position 28 is occupied byI, position 30 is occupied by T, position 38 is occupied by K, position48 is occupied by I, position 67 is occupied by K, position 68 isoccupied by A, position 70 is occupied by L, and/or position 95 isoccupied by F.
 3. The antibody or binding fragment of claim 1, whereinlight chain position 5 is occupied by S.
 4. The antibody or bindingfragment of claim 1, wherein the sequence of the heavy chain variableregion is selected from SEQ ID NOs. 13-25 (RHA through RHM); and thesequence of the light chain variable region is SEQ ID NO. 26 (RKA). 5.The antibody or binding fragment of claim 1, wherein the sequence of theheavy chain variable region is selected from SEQ ID NOs. 13-25 (RHAthrough RHM); and the sequence of the light chain variable region is SEQID NO. 27 (RKB).
 6. The antibody or binding fragment of claim 1, whereinthe sequence of the heavy chain variable region comprises SEQ ID NO. 14and the sequence of the light chain variable region comprises SEQ ID NO.26.
 7. The antibody or binding fragment of claim 1, wherein the sequenceof the heavy chain variable region comprises SEQ ID NO. 15 and thesequence of the light chain variable region comprises SEQ ID NO.
 26. 8.The antibody or binding fragment of claim 1, wherein the sequence of theheavy chain variable region comprises SEQ ID NO. 16 and the sequence ofthe light chain variable region comprises SEQ ID NO.
 26. 9. The antibodyor binding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 17 and the sequence of the lightchain variable region comprises SEQ ID NO.
 26. 10. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 18 and the sequence of the lightchain variable region comprises SEQ ID NO.
 26. 11. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 19 and the sequence of the lightchain variable region comprises SEQ ID NO.
 26. 12. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 20 and the sequence of the lightchain variable region comprises SEQ ID NO.
 26. 13. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 21 and the sequence of the lightchain variable region comprises SEQ ID NO.
 26. 14. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 22 and the sequence of the lightchain variable region comprises SEQ ID NO.
 26. 15. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 23 and the sequence of the lightchain variable region comprises SEQ ID NO.
 26. 16. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 24 and the sequence of the lightchain variable region comprises SEQ ID NO.
 26. 17. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 25 and the sequence of the lightchain variable region comprises SEQ ID NO.
 26. 18. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 13 and the sequence of the lightchain variable region comprises SEQ ID NO.
 27. 19. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 14 and the sequence of the lightchain variable region comprises SEQ ID NO.
 27. 20. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 15 and the sequence of the lightchain variable region comprises SEQ ID NO.
 27. 21. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 16 and the sequence of the lightchain variable region comprises SEQ ID NO.
 27. 22. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 17 and the sequence of the lightchain variable region comprises SEQ ID NO.
 27. 23. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 18 and the sequence of the lightchain variable region comprises SEQ ID NO.
 27. 24. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 19 and the sequence of the lightchain variable region comprises SEQ ID NO.
 27. 25. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 20 and the sequence of the lightchain variable region comprises SEQ ID NO.
 27. 26. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 21 and the sequence of the lightchain variable region comprises SEQ ID NO.
 27. 27. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 22 and the sequence of the lightchain variable region comprises SEQ ID NO.
 27. 28. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 23 and the sequence of the lightchain variable region comprises SEQ ID NO.
 27. 29. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 24 and the sequence of the lightchain variable region comprises SEQ ID NO.
 27. 30. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 25 and the sequence of the lightchain variable region comprises SEQ ID NO.
 27. 31. The antibody orbinding fragment of claim 1 wherein the antibody is of the IgG1 isotype.32. The antibody or binding fragment of claim 1 wherein the antibody isof the IgG4 isotype.
 33. The antibody or binding fragment of claim 1,wherein the sequence of the heavy chain variable region comprises SEQ IDNO. 14, the sequence of the light chain variable region comprises SEQ IDNO. 26, and the antibody is of the IgG1 isotype.
 34. The antibody orbinding fragment of claim 1, wherein the sequence of the heavy chainvariable region comprises SEQ ID NO. 16, the sequence of the light chainvariable region comprises SEQ ID NO. 26, and the antibody is of the IgG1isotype.
 35. The antibody or binding fragment of claim 1, wherein thesequence of the heavy chain variable region comprises SEQ ID NO. 17, thesequence of the light chain variable region comprises SEQ ID NO. 26, andthe antibody is of the IgG1 isotype.
 36. The antibody or bindingfragment of claim 1, wherein the sequence of the heavy chain variableregion comprises SEQ ID NO. 25, the sequence of the light chain variableregion comprises SEQ ID NO. 26, and the antibody is of the IgG1 isotype.37. The antibody or binding fragment of claim 1, wherein the sequence ofthe heavy chain variable region comprises SEQ ID NO. 16, the sequence ofthe light chain variable region comprises SEQ ID NO. 27, and theantibody is of the IgG1 isotype.
 38. The antibody or binding fragment ofclaim 1, wherein the sequence of the heavy chain variable regioncomprises SEQ ID NO. 17, the sequence of the light chain variable regioncomprises SEQ ID NO. 27, and the antibody is of the IgG1 isotype. 39.The antibody or binding fragment of claim 1, wherein the sequence of theheavy chain variable region comprises SEQ ID NO. 16, the sequence of thelight chain variable region comprises SEQ ID NO. 27, and the antibody isof the IgG4 isotype.
 40. The antibody or binding fragment of claim 1,wherein the sequence of the heavy chain variable region is comprises IDNO. 17, the sequence of the light chain variable region comprises SEQ IDNO. 27, and the antibody is of the IgG4 isotype.
 41. An antibody, or atau-binding fragment thereof, according to claim 1 wherein said antibodyor binding fragment binds at least one epitope selected from the groupconsisting of HQPGGG (SEQ ID NO: 148), HVPGGG (SEQ ID NO: 149), andHKPGGG (SEQ ID NO: 150).
 42. An immunoconjugate having the formula(A)-(L)-(C), wherein: (A) is an antibody or binding fragment thereof ofclaim 1; (L) is a linker; and (C) is an agent; and wherein said linker(L) links (A) to (C).
 43. A nucleic acid molecule encoding any of theantibodies or binding fragments of claim
 1. 44. A vector comprising anucleic acid according to claim
 43. 45. A host cell comprising a vectoraccording to claim
 44. 46. A method of producing an antibody ortau-binding fragment thereof that binds to human tau comprisingculturing the host cell of claim 45 so that the nucleic acid isexpressed and the antibody, tau-binding fragment, heavy chain, or lightchain thereof produced.
 47. The antibody or binding fragment of claim 1,wherein the sequence of the heavy chain variable region comprises SEQ IDNO. 13 and the sequence of the light chain variable region comprises SEQID NO.
 26. 48. The antibody or binding fragment of claim 47 wherein theantibody is of the IgG1 isotype.
 49. The antibody or binding fragment ofclaim 47 wherein the antibody is of the IgG4 isotype.
 50. Apharmaceutical composition comprising the antibody or binding fragmentaccording to claim 1 and a pharmaceutically acceptable carrier, diluent,excipient, or stabilizer.
 51. A method of treating Alzheimer's Diseaseor another tauopathy in a subject having Alzheimer's Disease or anothertauopathy, comprising administering to the subject a therapeuticallyeffective amount of (1) a composition comprising an antibody ortau-binding fragment according to claim 1, or (2) a compositionaccording to claim
 50. 52. A method of promoting clearance of tauaggregates from the brain of a subject having, suspected of having, orbeing prone to have Alzheimer's Disease or another tauopathy, comprisingadministering to the subject a therapeutically effective amount of (1)an antibody or tau-binding fragment according to claim 1, or (2) acomposition according to claim
 50. 53. A method of slowing progressionof AD or another tauopathy in a subject having Alzheimer's Disease oranother tauopathy, comprising administering to the subject atherapeutically effective amount of (1) an antibody or tau-bindingfragment according to claim 1, or (2) a composition according to claim50.
 54. A method of ameliorating the symptoms of AD or another tauopathyin a subject having Alzheimer's Disease or another tauopathy, comprisingadministering to the subject a therapeutically effective amount of (1)an antibody or tau-binding fragment according to claim 1, or (2) acomposition according to claim
 50. 55. A method of treating cognitiveimpairment in a subject having Alzheimer's Disease or another tauopathy,comprising administering to the subject a therapeutically effectiveamount of (1) an antibody or tau-binding fragment according to claim 1,or (2) a composition according to claim
 50. 56. A humanized anti-tauantibody, or a tau-binding fragment thereof, wherein said antibody orbinding fragment comprises: a. a heavy chain variable region comprisingCDR-H1, CDR-H2, and CDR-H3 of SEQ ID NOs. 1, 2, and 3, respectively, anda framework from human immunoglobulin M65092 (SEQ ID NO. 71); and b. alight chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of SEQID NOs. 4, 5, and 6, respectively, and a framework from humanimmunoglobulin X72449 (SEQ ID NO. 65); and c. heavy chain and lightchain constant regions from a human immunoglobulin.
 57. An anti-tauantibody, or a tau-binding fragment thereof, wherein said antibody orbinding fragment comprises: a. a heavy chain having the amino acidsequence of any one of SEQ ID NO. 28-40 and 43-55; and b. a light chainhaving the amino acid sequence of any one of SEQ ID NO. 57 and
 58. 58. Amethod of evaluating a subject having, suspected of having, or beingprone to have Alzheimer's Disease or another tauopathy, the methodcomprising the step of detecting binding of an antibody or tau-bindingfragment of claim 1 to a component of a biological sample from thesubject, wherein the detection of binding to the biological sample isindicative of Alzheimer's Disease or another tauopathy in the subject.